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Heavy metals in locus ceruleus and motor neurons in motor neuron disease

Heavy metals in locus ceruleus and motor neurons in motor neuron disease Background: The causes of sporadic amyotrophic lateral sclerosis (SALS) and other types of motor neuron disease (MND) remain largely unknown. Heavy metals have long been implicated in MND, and it has recently been shown that inorganic mercury selectively enters human locus ceruleus (LC) and motor neurons. We therefore used silver nitrate autometallography (AMG) to look for AMG-stainable heavy metals (inorganic mercury and bismuth) in LC and motor neurons of 24 patients with MND (18 with SALS and 6 with familial MND) and in the LC of 24 controls. Results: Heavy metals in neurons were found in significantly more MND patients than in controls when comparing: (1) the presence of any versus no heavy metal-containing LC neurons (MND 88%, controls 42%), (2) the median percentage of heavy metal-containing LC neurons (MND 9.5%, control 0.0%), and (3) numbers of individuals with heavy metal-containing LC neurons in the upper half of the percentage range (MND 75%, controls 25%). In MND patients, 67% of remaining spinal motor neurons contained heavy metals; smaller percentages were found in hypoglossal, nucleus ambiguus and oculomotor neurons, but none in cortical motor neurons. The majority of MND patients had heavy metals in both LC and spinal motor neurons. No glia or other neurons, including neuromelanin-containing neurons of the substantia nigra, contained stainable heavy metals. Conclusions: Uptake of heavy metals by LC and lower motor neurons appears to be fairly common in humans, though heavy metal staining in the LC, most likely due to inorganic mercury, was seen significantly more often in MND patients than in controls. The LC innervates many cell types that are affected in MND, and it is possible that MND is triggered by toxicant-induced interactions between LC and motor neurons. Keywords: Motor neuron disease, Amyotrophic lateral sclerosis, Toxicant, Heavy metal, Neurotoxin, Locus ceruleus, Motor neurons, Autometallography, Mercury, Sporadic ALS, Familial ALS Background The LC has many interactions with motor neurons so The causes of most cases of amyotrophic lateral sclerosis it is possible that some combination of toxicant uptake (ALS), the most common form of motor neuron disease by the LC and motor neurons might underlie MND. A (MND), as well as the other subtypes of MND, are still previous study of ours using the histochemical technique unknown [1]. So far only a modest proportion of spor- of autometallographic silver amplification (AMG) found adic MND patients have been found to harbour causa- heavy metals in spinal motor neurons in patients with tive genetic mutations, and attention has again turned to MND and controls, but the LC was not examined in that the possibility that in some patients the disease may be study [3]. We therefore used AMG to examine heavy caused by an environmental toxicant that affects motor metals in both LC and spinal motor neurons, using tis- neurons preferentially. The recent finding that one metal sue from previously-reported as well as from additional toxicant, inorganic mercury, enters the human locus cer- MND patients in whom paraffin blocks containing the uleus (LC) and motor neurons selectively after mercury LC were available. Updated histological criteria for heavy self-injection suggests this pathway could be used by metal staining and new quantitative procedures for the toxicants to enter and damage motor neurons [2]. presence of heavy metals in neurons were employed. The CNS was also sampled widely to see if heavy metal * Correspondence: roger.pamphlett@sydney.edu.au staining was specific for LC and motor neurons. Department of Pathology, The Stacey Motor Neuron Disease Laboratory, Sydney Medical School, The University of Sydney, Sydney, Australia © 2013 Pamphlett and Kum Jew; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 2 of 15 http://www.actaneurocomms.org/content/1/1/81 Methods MND, based on lower motor neuron loss, TDP-43 inclu- Nomenclature sions in remaining spinal motor neurons, and no increase (1) The term “toxicant” is used to describe a poison that is in microglia in the lateral corticospinal tracts on CD-68 put into the environment by human activity, in contrast to staining [5]. Familial MND was further classified into a toxin, which is a poison produced naturally by an organ- those with: (1) Mutations in SOD1 or the androgen recep- ism; the term toxicant does not necessarily imply damage tor gene. (2) Probable C9orf72 mutations, based on the to cells, since the toxicant may be at too low a concentra- characteristic p62 neuronal inclusions in the hippocampus tion to cause cell injury. (2) The term “heavy metal” in this or cerebellum [6]. (3) Unknown mutations, for patients study refers to the two metals that can be stained using who did not fit into the previous two categories. the routine AMG process, inorganic mercury and bis- muth. Although it seems most likely that the metal dem- Controls onstrated by AMG in this study is inorganic mercury Controls were 24 individuals (12 male, 12 female, age range (since exposure to bismuth is uncommon), we use the 24-86 years, mean age 59 years SD 20 years) in whom par- term metal toxicant or heavy metal instead of inorganic affin blocks containing the locus ceruleus were available, mercury since we cannot definitively identify the metal from the same sources and the same time period as the concerned using paraffin sections. (3) Unless otherwise MND blocks (Table 1). Controls consisted of: (1) Seventeen stated, the term “locus ceruleus” (LC) refers to the locus individuals who died from non-neurological conditions ceruleus nucleus in the pons, to prevent confusion with where no brain pathology was found. (2) Three individuals the nucleus of an individual LC neuron. who met the DSMIV criteria for alcohol abuse, but who had no brain pathology. (3) Three individuals with Parkin- Cases and controls son’s disease. (4) One individual with multiple sclerosis. In Cases addition to the control LC blocks, 6 controls had midbrain Cases were 24 patients (13 male, 11 female, age range 38– blocks containing the substantia nigra, 6 had spinal cord 84 years, mean age 61 years SD 13 years) with a clinical blocks, and 4 had medulla oblongata blocks. diagnosis of MND, made by a neurologist, where the diag- The project was approved by the Human Ethics Com- nosis was confirmed pathologically on post mortem exam- mittee of Sydney South West Area Health Service. ination of brain and spinal cord tissue performed between 1988 and 1999 (Table 1). MND cases were restricted to Autometallography those in whom available formalin-fixed paraffin-embedded 7 μm paraffin sections were stained for metal toxicants blocks contained: (1) Neuromelanin-containing neurons using silver nitrate autometallography (AMG), a silver amp- of the locus ceruleus in the rostral pons. (2) Anterior horn lification method which under routine conditions stains the motor neuron cell bodies in the spinal cord (lumbar and/ sulphides or selenides of mercury, silver, and bismuth [7,8]. or cervical). (3) Hypoglossal motor neurons in the medulla Briefly, paraffin sections were placed in physical developer oblongata. In MND patients tissue blocks were also containing 50% gum arabic, citrate buffer, hydroquinone available from the frontal motor cortex, which had been and silver nitrate at 26°C for 80 min in the dark, then sampled horizontally to include the maximum number washed in 5% sodium thiosulphate to remove unbound sil- of corticomotor neurons [4]), occipital cortex, midbrain, ver. Sections were counterstained with Harris hematoxylin hippocampus, caudate-putamen, and cerebellum. and viewed under bright-field illumination. Because Harris The results of neurological examinations were incom- haematoxylin contains a small amount of mercuric oxide, plete in some of the archival cases, though family histor- sections were also counterstained with mercury-free Im- ies of MND were available from all. During this period proved Harris haematoxylin, but no difference in AMG of tissue collection the only genetic tests available were staining was seen. Silver-coated metal deposits were seen as for mutations in the gene for superoxide-dismutase 1 black-staining grains. In each staining run, a positive (SOD1) and for repeat expansions in the androgen re- control section was included of mouse spinal cord ceptor gene for Kennedy’s disease, and no frozen tissue motor neurons which contained mercury deposits after was available for DNA extraction. We therefore immuno- an intraperitoneal injection of 2 μg/g mercuric chloride stained spinal cord sections with TDP-43 and CD-68, and [9]. Parallel sections of selected AMG-staining sections hippocampal and cerebellar sections with p62 and TDP- were pretreated for 2 h with 1% potassium cyanide to 43 to enable classification as: (1) Sporadic or familial distinguish any silver deposits, which disappear after MND (FMND), based on family history. (2) Classic ALS, this treatment [10]. based on upper and lower motor neuron loss, TDP-43 in- AMG staining of individual neurons was graded as: clusions in spinal motor neurons, and increased microglia (1) Negative, when there were no or fewer than 10 in the lateral corticospinal tracts on CD-68 staining. (3) AMG grains per neuron. (2) Light, when there were 10 Sporadic progressive muscular atrophy (SPMA) variant of or more AMG grains restricted to pigment-containing Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 3 of 15 http://www.actaneurocomms.org/content/1/1/81 Table 1 Details of heavy metal staining in LC and motor neurons of MND patients and controls Group Diagnosis Age Gender LC no. LC no. LC% LC% Pos SMN AMG 12n AMG NAm AMG 3n/4n AMG Pos Neg Pos Category grade grade grade grade MND SALS 71 Female 0 68 0 Low - - - 4n- SALS 62 Female 1 72 1 Low - - - 4n+ SALS 66 Female 2 90 2 Low + + - 4n- SALS 38 Male 4 81 5 High - - - NA SALS 77 Female 4 72 5 High + - - 4n+ SALS 81 Female 5 52 9 High ++ ++ ++ 3n+ SALS 59 Female 14 82 15 High - - - 4n- SALS 46 Male 12 60 17 High ++ + + 4n+ SALS 67 Female 17 66 20 High ++ ++ ++ 4n- SALS 45 Male 13 46 22 High - - ++ 4n- SALS C9orf72 74 Male 15 48 24 High - - - 4n- SALS 60 Female 32 79 29 High + + - 3n- SALS 49 Male 27 57 32 High + + ++ NA SPMA C9orf72 60 Female 0 60 0 Low + + - 4n- SPMA 58 Male 11 97 10 High ++ - - 4n+ SPMA 84 Male 21 64 25 High + - - 3n- SPMA 74 Male 23 48 32 High ++ - + 4n+ SPMA 53 Male 56 14 80 High + + - 4n- FMND 70 Male 21 75 22 High - - - 4n- FMND C9orf72 59 Male 2 32 6 High + - + 4n- FMND C9orf72 76 Female 5 79 6 High + + - 4n- FMND SOD1 41 Male 0 50 0 Low ++ - - 4n- FMND SOD1 47 Female 4 90 4 High ++ + ++ 4n- FMND Kennedy 53 Male 2 101 2 Low - - - 4n- Control Normal 86 Female 0 35 0 Low + + + NA Normal 55 Female 0 38 0 Low - - - NA Normal 77 Male 0 42 0 Low - NA NA NA Normal 24 Male 0 122 0 Low - NA NA NA Normal 82 Female 0 66 0 Low NA NA NA NA Normal 77 Female 0 29 0 Low NA NA NA NA Normal 44 Male 0 90 0 Low NA NA NA NA Normal 30 Female 0 82 0 Low NA NA NA NA Normal 75 Female 0 45 0 Low NA NA NA NA Normal 71 Male 1 74 1 Low NA NA NA NA Normal 47 Female 1 57 2 Low NA NA NA NA Normal 77 Female 2 98 2 Low NA NA NA NA Normal 55 Male 3 99 3 Low NA NA NA NA Normal 26 Male 3 54 5 High + + - NA Normal 52 Female 6 75 7 High NA NA NA NA Normal 54 Male 12 65 16 High NA NA NA NA Normal 74 Male 9 35 20 High - - - 4n+ Alcohol 36 Male 0 58 0 Low NA NA NA NA Alcohol 49 Female 0 70 0 Low NA NA NA NA Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 4 of 15 http://www.actaneurocomms.org/content/1/1/81 Table 1 Details of heavy metal staining in LC and motor neurons of MND patients and controls (Continued) Alcohol 33 Male 20 57 26 High NA NA NA NA Multiple sclerosis 51 Female 17 69 20 High NA NA NA NA Parkinson dis. 83 Male 0 13 0 Low + NA NA 3n++ Parkinson dis. 72 Female 0 12 0 Low NA NA NA 4n+ Parkinson dis. 74 Male 0 11 0 Low NA NA NA 4n- 3n: Oculomotor neurons, 4n: Trochlear neurons, 12n: Hypoglossal neurons, NA: Not available, NAm: Nucleus ambiguus neurons, SMN: Spinal motor neurons (see text for other abbreviations). (either neuromelanin or lipofuscin) regions of the neuron. by potassium cyanide pre-treatment, indicating the (3) Dense, when there were more than 10 AMG grains in staining was not due to silver deposits [10]. This indi- non-pigmented regions of the neuron, or when the grains cates that the standard AMG method used in this study were too compact to judge whether any underlying neur- demonstrated either mercury or bismuth in the cells onal pigment was present. [11,12]. A summary of heavy metal staining in each indi- vidual is shown in Table 1. Quantitation of locus ceruleus neurons containing AMG grains Heavy metals in the locus ceruleus To compare the percentage of AMG-stained LC neurons The density of neurons in cross-sections of the LC of between MND patients and controls, a 10×10 grid, with MND patients appeared similar to those of the normal right and lower exclusion margins, viewed at 400× mag- controls, as far as could be judged visually on one (vari- nification, was stepped sequentially through the LC. To able) level of the LC without formal quantitation. In the be included for quantitation, an AMG-stained LC neuron three Parkinson’s disease patients, however, LC neuronal was defined as any neuron (pigmented or non-pigmented) density was reduced by about 80% when compared to with a maximum diameter greater than 26 μm (the length normal control counts at the same horizontal level of of the side of one grid square) that contained 10 or more the pons, leaving only a few scattered surviving neurons. AMG grains. Neurons counted were those enclosed by an area bordered by the LC pigmented neurons. Large Heavy metals in LC neurons (greater than 26 μm in diameter) non-pigmented neu- Either no, light, or dense heavy metal staining could be seen rons made up about one-quarter of the neuronal count. in LC neurons (Figure 1). A mosaic of LC neurons contain- The numbers of neurons counted varies between cases ing no, light or dense heavy metal staining was usually seen. mostly because the area of the LC varies greatly through- LC neurons with heavy metals mostly contained neurome- out its length. A pilot study indicated that the percentage lanin granules as well. In some neurons dense heavy metal of AMG-stained LC neurons was similar between sides, staining made it difficult to judge whether or not the so only one randomly-selected side was counted. The neuron contained any neuromelanin (Figure 1). No glial quantifier (RP) was blinded to the diagnosis by having the cells within the LC contained heavy metals. label on each slide obscured. One slide counted on 10 dif- ferent occasions gave a coefficient of error of less than 5%. Comparison between heavy metal staining in MND and The small number of spinal motor neurons in SMND control LC neurons patients did not allow for formal quantitation of AMG Significantly more MND patients (88%) than controls staining. (42%) had an LC that contained at least one neuron with heavy metal staining (chi square p = 0.002) (Figure 2A). Statistics In addition, MND patients had significantly greater per- GraphPad Prism 5 was used to analyse contingency ta- centages of LC neurons with heavy metals than controls bles with Fisher’s exact test. Non-Gaussian continuous (MND median 9.5%, control median 0.0%, Mann–Whitney variable results were analysed with Mann-Whitney non- p = 0.0006) (Figure 2B). parametric tests. Double-sided p values were calculated In both MND patients and controls, some LC contained with alpha <0.05. very few heavy metal-stained neurons, which are likely to be insufficient in number to be of biological importance. Results Percentages of LC neurons containing heavy metals were Heavy metal staining in the brain and brain stem therefore divided into upper and lower halves, based on a The only CNS cells to stain for heavy metals were neu- median value for the whole cohort of 4% stained neurons rons in the LC and motor neurons in the spinal cord per individual. The low-percentage of AMG-stained neu- %AMG and brain stem. Heavy metal staining was not removed rons (low ) category had 0% to 3% heavy metal- Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 5 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 1 Heavy metal staining in the LC of a MND patient. (A) One LC neuron in this field stains densely with AMG, making it difficult to judge whether the neuromelanin is obscured or whether this is a non-pigmented neuron. (B) A pigmented neuron with light AMG staining, seen as more than 10 small black grains within the yellow-brown neuromelanin. (C) Three pigmented neurons with no quantifiable AMG grains (none in the two neurons on the right, and fewer than 10 grains in the neuron on the left). (D) A large non-pigmented neuron with no heavy metal staining. (E) Three small non-pigmented neurons with no heavy metal staining. No glial cells in the LC stain for heavy metals. Bar = 15 μm. AMG and hematoxylin. stained LC neurons per individual, and the high- particular in the sporadic progressive muscular atrophy %AMG %AMG %AMG percentage (high )category 4% or more heavy (SPMA) subgroup (Figure 3A). Low and high metal-stained LC neurons per individual. The LC of categories were seen in similar numbers in all three sub- MND patients were significantly more likely to be in groups (Figure 3B). %AMG the high category (75%) than those of controls All except 1 of the 13 SALS patients had LC nuclei in the %AMG (25%) (chi-square p = 0.0012) (Figure 2C). high category. The MND patient with the highest per- centage of heavy metal-stained LC neurons (80%) had SPMA. Comparison of heavy metal-stained LC neurons in Five of the 6 familial MND (FMND) patients had MND subtypes heavy metal-stained LC neurons, with 4 of these in the The percentage of heavy metal-stained LC neurons per %AMG high category. individual varied widely in all three MND subgroups, in Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 6 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 2 Comparison of LC heavy metal staining between MND patients and controls. (A) The number of MND patients having any heavy metal staining neurons in their LC was greater than in controls (chi square p = 0.002). (B) LC neurons containing heavy metal staining were present in both MND and control groups, but were significantly higher in the MND group (Mann–Whitney p = 0.0006). Bars = median values. (C) When the percentages of LC neurons containing heavy metals were divided into high (upper 50% of range) and low (lower 50% of range) categories, MND patients were predominantly in the high category, significantly different to the mirror-image result in controls (chi-square p = 0.0012). Heavy metals in LC neurons: the effects of age and gender To look for age effects on heavy metal staining in the LC, the cohort was divided into younger (24–59 years) and older (60–86 years) groups, based on a median age of 59.5 years. The group in the lower half of the age range had a slightly greater proportion of LC nuclei in %AMG the high category (58%) compared to the older group (42%), but this did not reach statistical signifi- cance (chi-square p = 0.39). Heavy metals did not there- fore appear to accumulate in the LC with age. The youngest individual to have AMG-stained LC neurons was a 26 year-old normal control individual, and the old- est an 84 year-old MND patient. %AMG More males (61%) had LC in the high category than females (39%), but this did not reach statistical sig- nificance (chi-square p = 0.25). Heavy metals in the LC in sporadic and familial MND Four out of 6 (67%) FMND patients, compared to 15 out of %AMG 18 (83%) sporadic MND patients, had LC in the high category. Therefore the majority of patients in both familial and sporadic MND groups had heavy metals in the LC in %AMG the high category (chi-square analysis not performed due to small numbers in the FMND group). No uniformity of LC AMG staining was present in SOD1 mutant or %AMG C9orf72 mutant MND patient groups, with both high %AMG and low categories within these groups. Heavy metals in motor neurons Spinal motor neurons All MND patients had severe losses of anterior horn spinal motor neurons, with fewer than 10 surviving neurons in some patients. No formal quantitation of heavy metal- stained spinal motor neurons could therefore be under- taken. Sixteen (67%) of the 24 MND patients had heavy metals in their spinal motor neurons; in 9 of these metal staining was light (Figure 4A), and in 7 dense (Figure 4B, C). Dense AMG staining was seen particularly in smaller neurons, with adjacent normal-sized neurons often showing no or only light AMG staining (Figure 4D). Heavy metals were seen in spinal motor neurons of all MND subgroups (SALS, SPMA and FMND). Three Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 7 of 15 http://www.actaneurocomms.org/content/1/1/81 Heavy metals in hypoglossal motor neurons All 12 individuals with hypoglossal motor neuron AMG staining (10 out of 24 MND patients, 2 out of 4 controls) also had AMG-stained spinal motor neurons (Figure 5A). On the other hand, 6 individuals who had AMG-stained spinal motor neurons had no hypoglossal motor neuron staining. Spinal motor neurons therefore appeared to take up metal toxicants more avidly than hypoglossal motor neurons. Heavy metals in nucleus ambiguus motor neurons Nucleus ambiguus neurons stained positively for heavy metals in 8 out of 22 SMND patients where the nucleus could be identified, and in 1 out of 4 controls (Figure 5B). Heavy metal staining in the nucleus ambiguus followed that in spinal motor neurons, apart from one SALS pa- tient who had positive nucleus ambiguus staining but negative staining in LC, spinal motor and hypoglossal motor neurons. Heavy metals in extraocular muscle motor neurons Heavy metal staining in extraocular motor neurons, ei- ther trochlear or oculomotor, was present in 6 (27%) of 22 MND patients (Figure 5C), and in 3 of 4 controls (2 of these having Parkinson’s disease). Heavy metals in cortical motor neurons No remaining cortical motor neurons contained AMG grains in any MND patient (Figure 5D). No glial AMG staining was present in the vicinity of these neurons. Heavy metals in other neurons An occasional neuromelanin-containing neuron in the medulla oblongata contained heavy metals, in individuals Figure 3 Comparison of LC heavy metal staining in SALS, SPMA in whom either AMG-stained LC or spinal motor neurons and FMND patients. (A) The percentage of LC neurons containing were also present. No heavy metal staining (not even a heavy metals varied widely in all MND subgroups, particularly in the single grain) was seen in pigmented or non-pigmented SMND group, but was not significantly different between groups. neurons of the substantia nigra (Figure 6A) in any individ- Bars = median values. (B) All MND subgroups had more high (upper ual, including the 3 patients with Parkinson’s disease. No 50% of range) than low (lower 50% of range) category LC neurons containing heavy metals, with no significant differences heavy metal staining was seen in any individual in neurons between groups. of the occipital cortex, hippocampus (Figure 6B), caudate- putamen, or cerebellum (Figure 6C). Neurons that con- tained abundant lipofuscin, such as those in the cerebellar dentate nucleus (Figure 6D) or the inferior olivary nucleus (54%) of the 7 controls had lightly AMG-stained spinal (Figure 6E), also contained no heavy metals. No heavy motor neurons. metals staining were seen in glial cells in any CNS region. Heavy metals in both LC and spinal motor neurons Discussion Heavy metals were present in both LC and spinal motor This study suggests that human LC neurons contain heavy neurons in 14 (58%) of the 24 MND patients. The ma- metals quite commonly. These LC heavy metals are, how- jority of MND patients with LC AMG staining there- ever, more likely to be found in patients with MND than fore also had spinal motor neuron AMG staining. Only controls. In the majority of MND patients, heavy metals 1 (17%) of the 6 control patients had both LC and were detected in spinal motor neurons as well. The AMG spinal motor AMG staining. silver amplification technique used detects the sulphides Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 8 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 4 Heavy metal staining in spinal motor neurons of MND patients (B, C and D from the same patient in Figure 1). (A) Light AMG staining can be seen within the lipofuscin of this normal-appearing spinal motor neuron, but none is present in the non-pigmented cytoplasm. Bar = 7 μm. (B) and (C) Two shrunken spinal motor neurons with dense AMG staining, which is either obscuring the lipofuscin, or is free in the cytoplasm. Bars = 20 μm. (D) A shrunken spinal motor neuron contains dense heavy metal staining (closed arrow), whereas an adjacent normal-sized neuron (open arrow) contains no heavy metal staining, despite containing lipofuscin. Bar = 15 μm. AMG and hematoxylin. or selenides of mercury, silver, and bismuth. After silver The locus ceruleus is a potential pathway for toxicants to was removed chemically from the tissues, AMG staining enter the brain was still present, indicating that either mercury and bis- Experimental animal studies show that circulating metal muth was likely to be causing the staining. Human expos- toxicants enter motor neurons selectively, probably via ure to bismuth is rare, occurring mostly after consumption retrograde axonal transport from the neuromuscular of bismuth-containing medications [12]. Therefore the junction [14]. However, only recently has it been shown metal seen on AMG in this study is most likely to be mer- that a heavy metal, inorganic mercury, enters LC neu- cury, a known neurotoxicant with numerous natural and rons selectively as well [2]. This entry into the LC prob- anthropogenic sources [13]. ably occurs because the LC innervates the great bulk of Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 9 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 5 Heavy metal staining in MND brain stem and cortical motor neurons. (A) Light AMG staining is present within the lipofuscin of three hypoglossal motor neurons. Bar = 15 μm. (B) These two nucleus ambiguus neurons contain light AMG staining. Bar = 20 μm. (C) Light AMG staining is seen within the lipofuscin of a trochlear nucleus neuron. Bar = 25 μm. (D) A shrunken cortical motor neuron (Betz cell, arrow) contains no AMG grains. Bar = 15 μm. AMG and hematoxylin. CNS microvessels [15], and so is exposed to a large vo- innervates a 20 meter length of capillary [18]. The LC con- lume of circulating blood. In fact, a rough calculation indi- tains numerous neurotransmitters in addition to noradren- cates that, with the number of normal human LC neurons aline [19], and re-uptake of these neurotransmitters at being 32,000 [16] and the total capillary length of the brain capillary terminals could be harnessed by a number of toxi- being 640 kilometres [17], each LC neuron on average cants to enter the LC via recycling mimicry [20]. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 10 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 6 Negative heavy metal staining in different CNS regions (all from the same MND patient in Figure 1 and Figure 5B, C and D). No heavy metal staining was present in: (A) Neuromelanin-containing neurons of the substantia nigra. Bar = 25 μm. (B) Neurons in the dentate gyrus of the hippocampus. Bar = 50 μm. (C) Purkinje cells (arrow) in the cerebellar cortex. Bar = 20 μm. (D) Lipofuscin-containing neurons in the cerebellar dentate nucleus. Bar = 25 μm. and (E) Lipofuscin-containing neurons in the inferior olivary nucleus. Bar = 30 μm. AMG and hematoxylin. Age and heavy metals in the locus ceruleus metals within his LC. One possible reason for this early In our previous study, we found no heavy metals in in- LC uptake of toxicant is that stressor-induced upregula- fant motor neurons, and suggested that metals were tion of the LC promotes bursts of toxicant intake into the likely to accumulate in motor neurons during aging [3]. nucleus [2]. In the present study, there was no correlation between increasing age and heavy metal content of the LC. This The amount of intracellular heavy metal varies widely raises the possibility that toxicant exposure in these individ- between adjacent locus ceruleus neurons uals was episodic, rather than continuous, in nature. Expos- A question that cannot be answered by this study was why ure may occur early in life, as evidenced by one 26 year-old only some LC neurons contained heavy metal staining, man without neurological disease already having heavy with densely-stained neurons usually found adjacent Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 11 of 15 http://www.actaneurocomms.org/content/1/1/81 to neurons with no AMG staining. This phenomenon was without the need for a toxicant to be within cortical also seen in a man who injected himself with a large dose motor neurons. After being activated by a stressor, LC of metallic mercury [2]. Topographic outputs of the ro- neurons supply noradrenaline directly to motor neurons, dent and primate LC to various brain regions have been as well as to microvessels and glial cells, all of which described, but not at the level of individual LC neurons. have noradrenaline surface receptors (Figure 7). Possibilities are that the toxicant-containing LC neurons Recent studies indicate that many CNS cells have nor- have a particularly large output to the capillary bed and so adrenaline receptors, so reduced noradrenaline output are exposed to greater amounts of circulating toxicant. can cause a number of deleterious changes to neurons Another possibility is that specific stressors, e.g., those re- [19,21]. These include changes that are implicated in quiring the LC to activate the motor system, result in the MND, such as blood–brain barrier permeability to toxi- uptake of toxicants into LC neurons that are activated to cants or inflammatory agents [22], decreased astrocytic deal with those specific stressors. function leading to increased synaptic glutamate [23], re- duced trophic support to the neuron [24], microglial ac- Could toxicants in the locus ceruleus trigger MND? tivation and inflammation [25], and oligodendrocyte We were not able to detect heavy metals in cortical dysfunction causing impaired myelination [26]. Finally, it motor neurons, despite these neurons being affected in has been suggested that the LC could transfer toxicants ALS, and being shown to contain mercury after expos- from the circulation to cortical motor neurons [2], since ure to metallic mercury [2]. This could be because of a the LC makes direct contact with both capillaries and survivor effect, with the toxicant-containing cortical motor neurons. A further discussion on how toxic dam- motor neurons dying early and leaving only toxicant-free age to the LC could result in different neurological con- motor neurons intact. Secondly, toxicants within the LC ditions has recently been published [18]. may reduce noradrenaline output to the cortical motor No technique currently exists to reliably measure the neurons and secondarily damage cortical motor neurons, concentration of toxicants such as heavy metals within Figure 7 Potential interactions between toxicant-containing LC and motor neurons (see text for details). Toxicant uptake into the LC could reduce noradrenaline output, resulting in: (A) A dysfunctional blood-brain barrier, which becomes permeable to the same or other toxicants, or to inflammatory agents. (B) Decreased astrocytic function leading to increased synaptic glutamate. (C) Reduced trophic support to the neuron. (D) Microglial activation and inflammation. (E) Oligodendrocyte dysfunction causing impaired myelination. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 12 of 15 http://www.actaneurocomms.org/content/1/1/81 individual neurons, which raises the question of how to as- Phenotypic variation in familial and sporadic MND: do sess how much heavy metal is in LC neurons. The lack of toxicants play a part? obvious neuronal loss in the metal-containing LCs of our Heavy metal-containing neurons were found in this MND patients could lead to the presumption that the study in familial, and not only sporadic, MND. One pos- amount of heavy metal was insufficient to damage the neu- sibility for this is that FMND-associated mutations in- rons. However, it has previously been shown that mouse crease the neuronal uptake of toxicants, possibly by spinal motor neurons that contain a non-toxic dose of inor- altering the permeability of the blood–brain barrier as ganic mercury suffer axonal shrinkage, without any loss of has been suggested for SOD1 [34], TARDBP and ANG numbers of motor neuron cell bodies [9]. This indicates mutations [35]. Other FMND mutations could damage that a low dose of a heavy metal can cause neuronal dam- mechanisms that protect neurons from toxicants, as has age without cell body loss, and by extrapolation suggests a been suggested for a number of MND genetic variants loss of noradrenaline from LC terminals could occur with- [36]. out the loss of toxicant-containing LC cell bodies. Of inter- The presence of toxicants within LC and motor neu- est, one of the few quantitative studies of the LC in MND rons could also explain the intra-familial variations in has shown neuronal shrinkage of LC cell bodies without age of disease onset and phenotype that is seen in cell loss [27], though no unbiased quantitative studies of FMND, as well as the inter-patient variations commonly cell numbers in the LC of MND patients have been re- found in sporadic MND. For example, we found that the ported. Even a moderate level of LC neuronal damage heavy metal content varied between hypoglossal and could have a deleterious effect on spinal motor neurons, spinal motor neurons, a possible reason why bulbar since the long LC axons extending down to the spinal cord symptoms can appear early, late or not at all in MND. would be particularly susceptible to toxic disturbances in their parent cell bodies [28]. Heavy metal staining in non-MND disease controls AMG can detect only a few heavy metals, but environ- With the recent finding of LC damage in multiple scler- mental exposures usually involve different types of toxi- osis [37] it is of interest that the LC of one multiple cants, often simultaneously. For example, cigarette smoke, sclerosis patient contained heavy metals. The heavy a possible risk factor for MND [29], contains 4,800 identi- metal staining within the LC of one individual with alco- fied compounds, including metal toxicants [30]. The heavy holism accords with the finding by some workers that metals identified in this study may not therefore be a LC neurons are damaged by alcohol excess [38]. cause of neurotoxicity, but may merely be a marker that indicates how readily a number of toxicants are entering Identification and quantification of heavy metals in neurons LC and motor neurons. Ideally, AMG should be followed up with some elemental Although we have suggested here that heavy metals in method to confirm the identity of the heavy metal in the the LC could play a part in the pathogenesis of MND, tissue. This has been achievable in humans exposed to an alternative explanation for the presence of heavy large amounts mercury or bismuth, where enough of the metals in the LC is that metals enter the LC after the metal was present in the tissues for quantitative methods start of the disease. Stress-induced uptake of toxicants to be of use [12]. However, AMG, being an amplification could take place after the onset of MND, because having technique, is more sensitive to the presence of toxicant a disease such as MND is likely to be highly stressful. metals than other currently available techniques of metal The finding of heavy metals in the LC could therefore detection (such as neutron activation analysis, proton- be a result of having the disease, rather than being a induced X-ray emission, atomic absorption spectropho- trigger for the disease. tometry, and electron emission X-ray spectrophotometry), though the lower amount of heavy metal that can be de- Heavy metal staining in extraocular muscle neurons tected with AMG has not been ascertained [10]. Unfortu- Extraocular muscles are affected late or not at all in MND nately, microprobe elemental analyses performed on [31]. Extraocular muscle neurons contained the least formalin-fixed paraffin sections are considered to be unre- heavy metal staining of all the motor neurons in the liable [39]. In our study, only a small proportion of the present study, raising the possibility that one reason they total number of cells in the pons, brain stem and spinal are spared is because they take up smaller toxicant loads. cord had AMG staining, which suggests only an in situ This may be because their neuromuscular junctions are microprobe technique, carried out on frozen sections, different to those of other muscles [32]. Curiously, 2 out would have a chance of detecting toxicants in the LC. of 3 Parkinson’s disease patients had heavy metals in their Motor neuron loss in most MND cases was too severe to extraocular muscle neurons; whether this is related to the be able to identify toxicants using elemental analysis. oculomotor problems associated with Parkinson’s disease AMG has been used extensively to study the distribu- [33] would require further study. tion of metal toxicants in experimental animals, used Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 13 of 15 http://www.actaneurocomms.org/content/1/1/81 occasionally in humans with known heavy metal exposure available was limited, since spinal cord tissue was usu- [2,12,40], but used only rarely in humans with no known ally removed at post mortem from controls only when toxicant exposure [11]. Humans differ from most non- clinically indicated, and LC-containing blocks were not primate animals in having extensive neuromelanin pigmen- available from all potential controls. (2) Only one (vari- tation in catecholaminergic neurons, as well as having able) level of the LC was available for examination, so lipofuscin in many aging neurons, including motor neu- we were unable to determine if differences in heavy rons [41]. It is therefore important to ensure that the metal content were present in rostral, middle and caudal AMG staining seen in human neurons is related to heavy parts of the LC; of note, cell loss in the LC in Alzheimer's metal staining, and not to some unknown reaction within and Parkinson's diseases has been found to vary along the pigmented neurons. The fact that no heavy metal staining length of the LC [45]. (3) We could not reliably detect was seen in any neuromelanin-containing neurons of the the subceruleus, which in primates is thought to inner- substantia nigra, or in other neurons containing high vate spinal motor neurons [46]. (4) No histories of toxi- levels of lipofuscin, indicates that these pigments them- cant exposure were available, so we do not know if any selves areunlikelytobethe source of theAMG staining. individuals had exposure to higher than normal levels of The heavy metals in this study localised preferentially mercury (e.g., living near an incinerator or a coal-burning to the neuromelanin of LC neurons, and to the lipofus- power station) or had been taking bismuth-containing cin of motor neurons, probably because these pigments medications. (5) No psychological, psychiatric, or stressful can bind metals [41]. Neuromelanin in the substantia life events histories were available, so we were unable to nigra of neurologically-normal individuals has an affin- determine levels of stress before the onset of MND. This ity for a number of physiologic and exogenous metal is of potential importance, since it has been suggested that ions, including mercury [42]. It has been suggested that stress, by activating the LC, could increase the uptake of the neuromelanin of the substantia nigra could play dif- toxicants into LC neurons [2]. (6) As noted above, we ferent roles in Parkinson’s disease, firstly as a protect- were unable to identify the type of heavy metal using par- ant by scavenging toxic substances such as exogenous affin sections, though this is likely to be inorganic mercury metals and pesticides, and later as a destructive agent since human exposure to bismuth is limited. when cell death releases the neuromelanin [43]. Neuro- melanin granules are probably not membrane-bound, Future studies and toxicants in unbound neuromelanin would be free The finding of potential disease-causing toxicants in a to interact with cellular constituents [41]. CNS region that is not morphologically damaged by Quantitation of the metal content of neuromelanin MND has implications for further neurotoxicological in- isolated from the substantia nigra of neurologically- vestigations into this disease. Attempts to locate toxicants normal individuals shows how neuromelanin can se- in MND tissues have generally been unsuccessful, prob- quester toxic metals that arise from environmental ably because toxicant-containing motor neurons have exposure, with many-fold accumulations of mercury largely disappeared by the time of death, and bulk ana- (1:96) and lead (1:1408) compared to surrounding tis- lysis of tissue is unlikely to detect toxicants affecting a sue (no corresponding data are available for LC neuro- small percentage of cells. An intact LC, however, is com- melanin) [44]. Of note, however, was the absence of pact and easily located, and would be an ideal candi- stainable heavy metals in any substantia nigra neurons date region for newer microanalysis methods that can in our study, despite the probability of age-related detect a range of toxicants [47]. heavy metal sequestration by neuromelanin in these The heavy metals within LC neurons in the present neurons. This suggests that the AMG staining we did study did not appear to cause structural damage to the see in the LC, especially in densely-stained neurons, neurons, and only minor changes in LC neuronal size represents a concentration of heavy metal that could have been reported in MND [27,48]. There are however be physiologically active. Indirect evidence of the po- a number of neurodegenerative and psychiatric disorders tential toxicity of the level of AMG staining seen is also where structural damage to the LC with associated nor- given by the findings from a man who injected himself adrenaline deficits have been described. Chief among with a large amount of metallic mercury [2], after these are Parkinson’s disease, Alzheimer’s disease, major which similar AMG staining of many LC neurons was depression and bipolar disorder [18,49,50]. In Alzheimer's seen, comparable in particular to the densely-stained disease it has been suggested that the LC is the first region neurons in the present study. of the brain to be involved by the disease, and at a very early age [51]. Environmental toxicants have been associ- Study limitations ated with all these disorders, and a search for toxicants Our study has a number of limitations: (1) The number within the LC of these patients could yield interesting of controls who had both LC and spinal cord tissue results. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 14 of 15 http://www.actaneurocomms.org/content/1/1/81 Conclusion 8. Danscher G, Stoltenberg M, Kemp K, Pamphlett R: Bismuth autometallography: protocol, specificity, and differentiation. J Histochem In conclusion, we have shown that the uptake of heavy Cytochem 2000, 48:1503–1510. metals by the human LC is a fairly common occurrence, 9. Pamphlett R, Png FY: Shrinkage of motor axons following systemic and is seen more in MND patients than controls. The exposure to inorganic mercury. J Neuropathol Exp Neurol 1998, 57:360–366. combination of toxicant uptake by the LC and lower 10. Danscher G, Rungby J: Differentiation of histochemically visualized motor neurons could result in damage to cortical and mercury and silver. Histochem J 1986, 18:109–114. lower motor neurons. Toxicant-induced LC damage with 11. Danscher G, Stoltenberg M, Juhl S: How to detect gold, silver and mercury in human brain and other tissues by autometallographic silver subsequent decreases of noradrenaline may explain the amplification. Neuropathol Appl Neuro 1994, 20:454–467. involvement in MND of multiple CNS regions and cell 12. Stoltenberg M, Hogenhuis JA, Hauw JJ, Danscher G: Autometallographic types. Further studies using newer methods of elemental tracing of bismuth in human brain autopsies. 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Submit your next manuscript to BioMed Central and take full advantage of: doi:10.1186/2051-5960-1-81 Cite this article as: Pamphlett and Kum Jew: Heavy metals in locus ceruleus and motor neurons in motor neuron disease. Acta • Convenient online submission Neuropathologica Communications 2013 1:81. • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Neuropathologica Communications Springer Journals

Heavy metals in locus ceruleus and motor neurons in motor neuron disease

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Abstract

Background: The causes of sporadic amyotrophic lateral sclerosis (SALS) and other types of motor neuron disease (MND) remain largely unknown. Heavy metals have long been implicated in MND, and it has recently been shown that inorganic mercury selectively enters human locus ceruleus (LC) and motor neurons. We therefore used silver nitrate autometallography (AMG) to look for AMG-stainable heavy metals (inorganic mercury and bismuth) in LC and motor neurons of 24 patients with MND (18 with SALS and 6 with familial MND) and in the LC of 24 controls. Results: Heavy metals in neurons were found in significantly more MND patients than in controls when comparing: (1) the presence of any versus no heavy metal-containing LC neurons (MND 88%, controls 42%), (2) the median percentage of heavy metal-containing LC neurons (MND 9.5%, control 0.0%), and (3) numbers of individuals with heavy metal-containing LC neurons in the upper half of the percentage range (MND 75%, controls 25%). In MND patients, 67% of remaining spinal motor neurons contained heavy metals; smaller percentages were found in hypoglossal, nucleus ambiguus and oculomotor neurons, but none in cortical motor neurons. The majority of MND patients had heavy metals in both LC and spinal motor neurons. No glia or other neurons, including neuromelanin-containing neurons of the substantia nigra, contained stainable heavy metals. Conclusions: Uptake of heavy metals by LC and lower motor neurons appears to be fairly common in humans, though heavy metal staining in the LC, most likely due to inorganic mercury, was seen significantly more often in MND patients than in controls. The LC innervates many cell types that are affected in MND, and it is possible that MND is triggered by toxicant-induced interactions between LC and motor neurons. Keywords: Motor neuron disease, Amyotrophic lateral sclerosis, Toxicant, Heavy metal, Neurotoxin, Locus ceruleus, Motor neurons, Autometallography, Mercury, Sporadic ALS, Familial ALS Background The LC has many interactions with motor neurons so The causes of most cases of amyotrophic lateral sclerosis it is possible that some combination of toxicant uptake (ALS), the most common form of motor neuron disease by the LC and motor neurons might underlie MND. A (MND), as well as the other subtypes of MND, are still previous study of ours using the histochemical technique unknown [1]. So far only a modest proportion of spor- of autometallographic silver amplification (AMG) found adic MND patients have been found to harbour causa- heavy metals in spinal motor neurons in patients with tive genetic mutations, and attention has again turned to MND and controls, but the LC was not examined in that the possibility that in some patients the disease may be study [3]. We therefore used AMG to examine heavy caused by an environmental toxicant that affects motor metals in both LC and spinal motor neurons, using tis- neurons preferentially. The recent finding that one metal sue from previously-reported as well as from additional toxicant, inorganic mercury, enters the human locus cer- MND patients in whom paraffin blocks containing the uleus (LC) and motor neurons selectively after mercury LC were available. Updated histological criteria for heavy self-injection suggests this pathway could be used by metal staining and new quantitative procedures for the toxicants to enter and damage motor neurons [2]. presence of heavy metals in neurons were employed. The CNS was also sampled widely to see if heavy metal * Correspondence: roger.pamphlett@sydney.edu.au staining was specific for LC and motor neurons. Department of Pathology, The Stacey Motor Neuron Disease Laboratory, Sydney Medical School, The University of Sydney, Sydney, Australia © 2013 Pamphlett and Kum Jew; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 2 of 15 http://www.actaneurocomms.org/content/1/1/81 Methods MND, based on lower motor neuron loss, TDP-43 inclu- Nomenclature sions in remaining spinal motor neurons, and no increase (1) The term “toxicant” is used to describe a poison that is in microglia in the lateral corticospinal tracts on CD-68 put into the environment by human activity, in contrast to staining [5]. Familial MND was further classified into a toxin, which is a poison produced naturally by an organ- those with: (1) Mutations in SOD1 or the androgen recep- ism; the term toxicant does not necessarily imply damage tor gene. (2) Probable C9orf72 mutations, based on the to cells, since the toxicant may be at too low a concentra- characteristic p62 neuronal inclusions in the hippocampus tion to cause cell injury. (2) The term “heavy metal” in this or cerebellum [6]. (3) Unknown mutations, for patients study refers to the two metals that can be stained using who did not fit into the previous two categories. the routine AMG process, inorganic mercury and bis- muth. Although it seems most likely that the metal dem- Controls onstrated by AMG in this study is inorganic mercury Controls were 24 individuals (12 male, 12 female, age range (since exposure to bismuth is uncommon), we use the 24-86 years, mean age 59 years SD 20 years) in whom par- term metal toxicant or heavy metal instead of inorganic affin blocks containing the locus ceruleus were available, mercury since we cannot definitively identify the metal from the same sources and the same time period as the concerned using paraffin sections. (3) Unless otherwise MND blocks (Table 1). Controls consisted of: (1) Seventeen stated, the term “locus ceruleus” (LC) refers to the locus individuals who died from non-neurological conditions ceruleus nucleus in the pons, to prevent confusion with where no brain pathology was found. (2) Three individuals the nucleus of an individual LC neuron. who met the DSMIV criteria for alcohol abuse, but who had no brain pathology. (3) Three individuals with Parkin- Cases and controls son’s disease. (4) One individual with multiple sclerosis. In Cases addition to the control LC blocks, 6 controls had midbrain Cases were 24 patients (13 male, 11 female, age range 38– blocks containing the substantia nigra, 6 had spinal cord 84 years, mean age 61 years SD 13 years) with a clinical blocks, and 4 had medulla oblongata blocks. diagnosis of MND, made by a neurologist, where the diag- The project was approved by the Human Ethics Com- nosis was confirmed pathologically on post mortem exam- mittee of Sydney South West Area Health Service. ination of brain and spinal cord tissue performed between 1988 and 1999 (Table 1). MND cases were restricted to Autometallography those in whom available formalin-fixed paraffin-embedded 7 μm paraffin sections were stained for metal toxicants blocks contained: (1) Neuromelanin-containing neurons using silver nitrate autometallography (AMG), a silver amp- of the locus ceruleus in the rostral pons. (2) Anterior horn lification method which under routine conditions stains the motor neuron cell bodies in the spinal cord (lumbar and/ sulphides or selenides of mercury, silver, and bismuth [7,8]. or cervical). (3) Hypoglossal motor neurons in the medulla Briefly, paraffin sections were placed in physical developer oblongata. In MND patients tissue blocks were also containing 50% gum arabic, citrate buffer, hydroquinone available from the frontal motor cortex, which had been and silver nitrate at 26°C for 80 min in the dark, then sampled horizontally to include the maximum number washed in 5% sodium thiosulphate to remove unbound sil- of corticomotor neurons [4]), occipital cortex, midbrain, ver. Sections were counterstained with Harris hematoxylin hippocampus, caudate-putamen, and cerebellum. and viewed under bright-field illumination. Because Harris The results of neurological examinations were incom- haematoxylin contains a small amount of mercuric oxide, plete in some of the archival cases, though family histor- sections were also counterstained with mercury-free Im- ies of MND were available from all. During this period proved Harris haematoxylin, but no difference in AMG of tissue collection the only genetic tests available were staining was seen. Silver-coated metal deposits were seen as for mutations in the gene for superoxide-dismutase 1 black-staining grains. In each staining run, a positive (SOD1) and for repeat expansions in the androgen re- control section was included of mouse spinal cord ceptor gene for Kennedy’s disease, and no frozen tissue motor neurons which contained mercury deposits after was available for DNA extraction. We therefore immuno- an intraperitoneal injection of 2 μg/g mercuric chloride stained spinal cord sections with TDP-43 and CD-68, and [9]. Parallel sections of selected AMG-staining sections hippocampal and cerebellar sections with p62 and TDP- were pretreated for 2 h with 1% potassium cyanide to 43 to enable classification as: (1) Sporadic or familial distinguish any silver deposits, which disappear after MND (FMND), based on family history. (2) Classic ALS, this treatment [10]. based on upper and lower motor neuron loss, TDP-43 in- AMG staining of individual neurons was graded as: clusions in spinal motor neurons, and increased microglia (1) Negative, when there were no or fewer than 10 in the lateral corticospinal tracts on CD-68 staining. (3) AMG grains per neuron. (2) Light, when there were 10 Sporadic progressive muscular atrophy (SPMA) variant of or more AMG grains restricted to pigment-containing Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 3 of 15 http://www.actaneurocomms.org/content/1/1/81 Table 1 Details of heavy metal staining in LC and motor neurons of MND patients and controls Group Diagnosis Age Gender LC no. LC no. LC% LC% Pos SMN AMG 12n AMG NAm AMG 3n/4n AMG Pos Neg Pos Category grade grade grade grade MND SALS 71 Female 0 68 0 Low - - - 4n- SALS 62 Female 1 72 1 Low - - - 4n+ SALS 66 Female 2 90 2 Low + + - 4n- SALS 38 Male 4 81 5 High - - - NA SALS 77 Female 4 72 5 High + - - 4n+ SALS 81 Female 5 52 9 High ++ ++ ++ 3n+ SALS 59 Female 14 82 15 High - - - 4n- SALS 46 Male 12 60 17 High ++ + + 4n+ SALS 67 Female 17 66 20 High ++ ++ ++ 4n- SALS 45 Male 13 46 22 High - - ++ 4n- SALS C9orf72 74 Male 15 48 24 High - - - 4n- SALS 60 Female 32 79 29 High + + - 3n- SALS 49 Male 27 57 32 High + + ++ NA SPMA C9orf72 60 Female 0 60 0 Low + + - 4n- SPMA 58 Male 11 97 10 High ++ - - 4n+ SPMA 84 Male 21 64 25 High + - - 3n- SPMA 74 Male 23 48 32 High ++ - + 4n+ SPMA 53 Male 56 14 80 High + + - 4n- FMND 70 Male 21 75 22 High - - - 4n- FMND C9orf72 59 Male 2 32 6 High + - + 4n- FMND C9orf72 76 Female 5 79 6 High + + - 4n- FMND SOD1 41 Male 0 50 0 Low ++ - - 4n- FMND SOD1 47 Female 4 90 4 High ++ + ++ 4n- FMND Kennedy 53 Male 2 101 2 Low - - - 4n- Control Normal 86 Female 0 35 0 Low + + + NA Normal 55 Female 0 38 0 Low - - - NA Normal 77 Male 0 42 0 Low - NA NA NA Normal 24 Male 0 122 0 Low - NA NA NA Normal 82 Female 0 66 0 Low NA NA NA NA Normal 77 Female 0 29 0 Low NA NA NA NA Normal 44 Male 0 90 0 Low NA NA NA NA Normal 30 Female 0 82 0 Low NA NA NA NA Normal 75 Female 0 45 0 Low NA NA NA NA Normal 71 Male 1 74 1 Low NA NA NA NA Normal 47 Female 1 57 2 Low NA NA NA NA Normal 77 Female 2 98 2 Low NA NA NA NA Normal 55 Male 3 99 3 Low NA NA NA NA Normal 26 Male 3 54 5 High + + - NA Normal 52 Female 6 75 7 High NA NA NA NA Normal 54 Male 12 65 16 High NA NA NA NA Normal 74 Male 9 35 20 High - - - 4n+ Alcohol 36 Male 0 58 0 Low NA NA NA NA Alcohol 49 Female 0 70 0 Low NA NA NA NA Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 4 of 15 http://www.actaneurocomms.org/content/1/1/81 Table 1 Details of heavy metal staining in LC and motor neurons of MND patients and controls (Continued) Alcohol 33 Male 20 57 26 High NA NA NA NA Multiple sclerosis 51 Female 17 69 20 High NA NA NA NA Parkinson dis. 83 Male 0 13 0 Low + NA NA 3n++ Parkinson dis. 72 Female 0 12 0 Low NA NA NA 4n+ Parkinson dis. 74 Male 0 11 0 Low NA NA NA 4n- 3n: Oculomotor neurons, 4n: Trochlear neurons, 12n: Hypoglossal neurons, NA: Not available, NAm: Nucleus ambiguus neurons, SMN: Spinal motor neurons (see text for other abbreviations). (either neuromelanin or lipofuscin) regions of the neuron. by potassium cyanide pre-treatment, indicating the (3) Dense, when there were more than 10 AMG grains in staining was not due to silver deposits [10]. This indi- non-pigmented regions of the neuron, or when the grains cates that the standard AMG method used in this study were too compact to judge whether any underlying neur- demonstrated either mercury or bismuth in the cells onal pigment was present. [11,12]. A summary of heavy metal staining in each indi- vidual is shown in Table 1. Quantitation of locus ceruleus neurons containing AMG grains Heavy metals in the locus ceruleus To compare the percentage of AMG-stained LC neurons The density of neurons in cross-sections of the LC of between MND patients and controls, a 10×10 grid, with MND patients appeared similar to those of the normal right and lower exclusion margins, viewed at 400× mag- controls, as far as could be judged visually on one (vari- nification, was stepped sequentially through the LC. To able) level of the LC without formal quantitation. In the be included for quantitation, an AMG-stained LC neuron three Parkinson’s disease patients, however, LC neuronal was defined as any neuron (pigmented or non-pigmented) density was reduced by about 80% when compared to with a maximum diameter greater than 26 μm (the length normal control counts at the same horizontal level of of the side of one grid square) that contained 10 or more the pons, leaving only a few scattered surviving neurons. AMG grains. Neurons counted were those enclosed by an area bordered by the LC pigmented neurons. Large Heavy metals in LC neurons (greater than 26 μm in diameter) non-pigmented neu- Either no, light, or dense heavy metal staining could be seen rons made up about one-quarter of the neuronal count. in LC neurons (Figure 1). A mosaic of LC neurons contain- The numbers of neurons counted varies between cases ing no, light or dense heavy metal staining was usually seen. mostly because the area of the LC varies greatly through- LC neurons with heavy metals mostly contained neurome- out its length. A pilot study indicated that the percentage lanin granules as well. In some neurons dense heavy metal of AMG-stained LC neurons was similar between sides, staining made it difficult to judge whether or not the so only one randomly-selected side was counted. The neuron contained any neuromelanin (Figure 1). No glial quantifier (RP) was blinded to the diagnosis by having the cells within the LC contained heavy metals. label on each slide obscured. One slide counted on 10 dif- ferent occasions gave a coefficient of error of less than 5%. Comparison between heavy metal staining in MND and The small number of spinal motor neurons in SMND control LC neurons patients did not allow for formal quantitation of AMG Significantly more MND patients (88%) than controls staining. (42%) had an LC that contained at least one neuron with heavy metal staining (chi square p = 0.002) (Figure 2A). Statistics In addition, MND patients had significantly greater per- GraphPad Prism 5 was used to analyse contingency ta- centages of LC neurons with heavy metals than controls bles with Fisher’s exact test. Non-Gaussian continuous (MND median 9.5%, control median 0.0%, Mann–Whitney variable results were analysed with Mann-Whitney non- p = 0.0006) (Figure 2B). parametric tests. Double-sided p values were calculated In both MND patients and controls, some LC contained with alpha <0.05. very few heavy metal-stained neurons, which are likely to be insufficient in number to be of biological importance. Results Percentages of LC neurons containing heavy metals were Heavy metal staining in the brain and brain stem therefore divided into upper and lower halves, based on a The only CNS cells to stain for heavy metals were neu- median value for the whole cohort of 4% stained neurons rons in the LC and motor neurons in the spinal cord per individual. The low-percentage of AMG-stained neu- %AMG and brain stem. Heavy metal staining was not removed rons (low ) category had 0% to 3% heavy metal- Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 5 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 1 Heavy metal staining in the LC of a MND patient. (A) One LC neuron in this field stains densely with AMG, making it difficult to judge whether the neuromelanin is obscured or whether this is a non-pigmented neuron. (B) A pigmented neuron with light AMG staining, seen as more than 10 small black grains within the yellow-brown neuromelanin. (C) Three pigmented neurons with no quantifiable AMG grains (none in the two neurons on the right, and fewer than 10 grains in the neuron on the left). (D) A large non-pigmented neuron with no heavy metal staining. (E) Three small non-pigmented neurons with no heavy metal staining. No glial cells in the LC stain for heavy metals. Bar = 15 μm. AMG and hematoxylin. stained LC neurons per individual, and the high- particular in the sporadic progressive muscular atrophy %AMG %AMG %AMG percentage (high )category 4% or more heavy (SPMA) subgroup (Figure 3A). Low and high metal-stained LC neurons per individual. The LC of categories were seen in similar numbers in all three sub- MND patients were significantly more likely to be in groups (Figure 3B). %AMG the high category (75%) than those of controls All except 1 of the 13 SALS patients had LC nuclei in the %AMG (25%) (chi-square p = 0.0012) (Figure 2C). high category. The MND patient with the highest per- centage of heavy metal-stained LC neurons (80%) had SPMA. Comparison of heavy metal-stained LC neurons in Five of the 6 familial MND (FMND) patients had MND subtypes heavy metal-stained LC neurons, with 4 of these in the The percentage of heavy metal-stained LC neurons per %AMG high category. individual varied widely in all three MND subgroups, in Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 6 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 2 Comparison of LC heavy metal staining between MND patients and controls. (A) The number of MND patients having any heavy metal staining neurons in their LC was greater than in controls (chi square p = 0.002). (B) LC neurons containing heavy metal staining were present in both MND and control groups, but were significantly higher in the MND group (Mann–Whitney p = 0.0006). Bars = median values. (C) When the percentages of LC neurons containing heavy metals were divided into high (upper 50% of range) and low (lower 50% of range) categories, MND patients were predominantly in the high category, significantly different to the mirror-image result in controls (chi-square p = 0.0012). Heavy metals in LC neurons: the effects of age and gender To look for age effects on heavy metal staining in the LC, the cohort was divided into younger (24–59 years) and older (60–86 years) groups, based on a median age of 59.5 years. The group in the lower half of the age range had a slightly greater proportion of LC nuclei in %AMG the high category (58%) compared to the older group (42%), but this did not reach statistical signifi- cance (chi-square p = 0.39). Heavy metals did not there- fore appear to accumulate in the LC with age. The youngest individual to have AMG-stained LC neurons was a 26 year-old normal control individual, and the old- est an 84 year-old MND patient. %AMG More males (61%) had LC in the high category than females (39%), but this did not reach statistical sig- nificance (chi-square p = 0.25). Heavy metals in the LC in sporadic and familial MND Four out of 6 (67%) FMND patients, compared to 15 out of %AMG 18 (83%) sporadic MND patients, had LC in the high category. Therefore the majority of patients in both familial and sporadic MND groups had heavy metals in the LC in %AMG the high category (chi-square analysis not performed due to small numbers in the FMND group). No uniformity of LC AMG staining was present in SOD1 mutant or %AMG C9orf72 mutant MND patient groups, with both high %AMG and low categories within these groups. Heavy metals in motor neurons Spinal motor neurons All MND patients had severe losses of anterior horn spinal motor neurons, with fewer than 10 surviving neurons in some patients. No formal quantitation of heavy metal- stained spinal motor neurons could therefore be under- taken. Sixteen (67%) of the 24 MND patients had heavy metals in their spinal motor neurons; in 9 of these metal staining was light (Figure 4A), and in 7 dense (Figure 4B, C). Dense AMG staining was seen particularly in smaller neurons, with adjacent normal-sized neurons often showing no or only light AMG staining (Figure 4D). Heavy metals were seen in spinal motor neurons of all MND subgroups (SALS, SPMA and FMND). Three Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 7 of 15 http://www.actaneurocomms.org/content/1/1/81 Heavy metals in hypoglossal motor neurons All 12 individuals with hypoglossal motor neuron AMG staining (10 out of 24 MND patients, 2 out of 4 controls) also had AMG-stained spinal motor neurons (Figure 5A). On the other hand, 6 individuals who had AMG-stained spinal motor neurons had no hypoglossal motor neuron staining. Spinal motor neurons therefore appeared to take up metal toxicants more avidly than hypoglossal motor neurons. Heavy metals in nucleus ambiguus motor neurons Nucleus ambiguus neurons stained positively for heavy metals in 8 out of 22 SMND patients where the nucleus could be identified, and in 1 out of 4 controls (Figure 5B). Heavy metal staining in the nucleus ambiguus followed that in spinal motor neurons, apart from one SALS pa- tient who had positive nucleus ambiguus staining but negative staining in LC, spinal motor and hypoglossal motor neurons. Heavy metals in extraocular muscle motor neurons Heavy metal staining in extraocular motor neurons, ei- ther trochlear or oculomotor, was present in 6 (27%) of 22 MND patients (Figure 5C), and in 3 of 4 controls (2 of these having Parkinson’s disease). Heavy metals in cortical motor neurons No remaining cortical motor neurons contained AMG grains in any MND patient (Figure 5D). No glial AMG staining was present in the vicinity of these neurons. Heavy metals in other neurons An occasional neuromelanin-containing neuron in the medulla oblongata contained heavy metals, in individuals Figure 3 Comparison of LC heavy metal staining in SALS, SPMA in whom either AMG-stained LC or spinal motor neurons and FMND patients. (A) The percentage of LC neurons containing were also present. No heavy metal staining (not even a heavy metals varied widely in all MND subgroups, particularly in the single grain) was seen in pigmented or non-pigmented SMND group, but was not significantly different between groups. neurons of the substantia nigra (Figure 6A) in any individ- Bars = median values. (B) All MND subgroups had more high (upper ual, including the 3 patients with Parkinson’s disease. No 50% of range) than low (lower 50% of range) category LC neurons containing heavy metals, with no significant differences heavy metal staining was seen in any individual in neurons between groups. of the occipital cortex, hippocampus (Figure 6B), caudate- putamen, or cerebellum (Figure 6C). Neurons that con- tained abundant lipofuscin, such as those in the cerebellar dentate nucleus (Figure 6D) or the inferior olivary nucleus (54%) of the 7 controls had lightly AMG-stained spinal (Figure 6E), also contained no heavy metals. No heavy motor neurons. metals staining were seen in glial cells in any CNS region. Heavy metals in both LC and spinal motor neurons Discussion Heavy metals were present in both LC and spinal motor This study suggests that human LC neurons contain heavy neurons in 14 (58%) of the 24 MND patients. The ma- metals quite commonly. These LC heavy metals are, how- jority of MND patients with LC AMG staining there- ever, more likely to be found in patients with MND than fore also had spinal motor neuron AMG staining. Only controls. In the majority of MND patients, heavy metals 1 (17%) of the 6 control patients had both LC and were detected in spinal motor neurons as well. The AMG spinal motor AMG staining. silver amplification technique used detects the sulphides Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 8 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 4 Heavy metal staining in spinal motor neurons of MND patients (B, C and D from the same patient in Figure 1). (A) Light AMG staining can be seen within the lipofuscin of this normal-appearing spinal motor neuron, but none is present in the non-pigmented cytoplasm. Bar = 7 μm. (B) and (C) Two shrunken spinal motor neurons with dense AMG staining, which is either obscuring the lipofuscin, or is free in the cytoplasm. Bars = 20 μm. (D) A shrunken spinal motor neuron contains dense heavy metal staining (closed arrow), whereas an adjacent normal-sized neuron (open arrow) contains no heavy metal staining, despite containing lipofuscin. Bar = 15 μm. AMG and hematoxylin. or selenides of mercury, silver, and bismuth. After silver The locus ceruleus is a potential pathway for toxicants to was removed chemically from the tissues, AMG staining enter the brain was still present, indicating that either mercury and bis- Experimental animal studies show that circulating metal muth was likely to be causing the staining. Human expos- toxicants enter motor neurons selectively, probably via ure to bismuth is rare, occurring mostly after consumption retrograde axonal transport from the neuromuscular of bismuth-containing medications [12]. Therefore the junction [14]. However, only recently has it been shown metal seen on AMG in this study is most likely to be mer- that a heavy metal, inorganic mercury, enters LC neu- cury, a known neurotoxicant with numerous natural and rons selectively as well [2]. This entry into the LC prob- anthropogenic sources [13]. ably occurs because the LC innervates the great bulk of Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 9 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 5 Heavy metal staining in MND brain stem and cortical motor neurons. (A) Light AMG staining is present within the lipofuscin of three hypoglossal motor neurons. Bar = 15 μm. (B) These two nucleus ambiguus neurons contain light AMG staining. Bar = 20 μm. (C) Light AMG staining is seen within the lipofuscin of a trochlear nucleus neuron. Bar = 25 μm. (D) A shrunken cortical motor neuron (Betz cell, arrow) contains no AMG grains. Bar = 15 μm. AMG and hematoxylin. CNS microvessels [15], and so is exposed to a large vo- innervates a 20 meter length of capillary [18]. The LC con- lume of circulating blood. In fact, a rough calculation indi- tains numerous neurotransmitters in addition to noradren- cates that, with the number of normal human LC neurons aline [19], and re-uptake of these neurotransmitters at being 32,000 [16] and the total capillary length of the brain capillary terminals could be harnessed by a number of toxi- being 640 kilometres [17], each LC neuron on average cants to enter the LC via recycling mimicry [20]. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 10 of 15 http://www.actaneurocomms.org/content/1/1/81 Figure 6 Negative heavy metal staining in different CNS regions (all from the same MND patient in Figure 1 and Figure 5B, C and D). No heavy metal staining was present in: (A) Neuromelanin-containing neurons of the substantia nigra. Bar = 25 μm. (B) Neurons in the dentate gyrus of the hippocampus. Bar = 50 μm. (C) Purkinje cells (arrow) in the cerebellar cortex. Bar = 20 μm. (D) Lipofuscin-containing neurons in the cerebellar dentate nucleus. Bar = 25 μm. and (E) Lipofuscin-containing neurons in the inferior olivary nucleus. Bar = 30 μm. AMG and hematoxylin. Age and heavy metals in the locus ceruleus metals within his LC. One possible reason for this early In our previous study, we found no heavy metals in in- LC uptake of toxicant is that stressor-induced upregula- fant motor neurons, and suggested that metals were tion of the LC promotes bursts of toxicant intake into the likely to accumulate in motor neurons during aging [3]. nucleus [2]. In the present study, there was no correlation between increasing age and heavy metal content of the LC. This The amount of intracellular heavy metal varies widely raises the possibility that toxicant exposure in these individ- between adjacent locus ceruleus neurons uals was episodic, rather than continuous, in nature. Expos- A question that cannot be answered by this study was why ure may occur early in life, as evidenced by one 26 year-old only some LC neurons contained heavy metal staining, man without neurological disease already having heavy with densely-stained neurons usually found adjacent Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 11 of 15 http://www.actaneurocomms.org/content/1/1/81 to neurons with no AMG staining. This phenomenon was without the need for a toxicant to be within cortical also seen in a man who injected himself with a large dose motor neurons. After being activated by a stressor, LC of metallic mercury [2]. Topographic outputs of the ro- neurons supply noradrenaline directly to motor neurons, dent and primate LC to various brain regions have been as well as to microvessels and glial cells, all of which described, but not at the level of individual LC neurons. have noradrenaline surface receptors (Figure 7). Possibilities are that the toxicant-containing LC neurons Recent studies indicate that many CNS cells have nor- have a particularly large output to the capillary bed and so adrenaline receptors, so reduced noradrenaline output are exposed to greater amounts of circulating toxicant. can cause a number of deleterious changes to neurons Another possibility is that specific stressors, e.g., those re- [19,21]. These include changes that are implicated in quiring the LC to activate the motor system, result in the MND, such as blood–brain barrier permeability to toxi- uptake of toxicants into LC neurons that are activated to cants or inflammatory agents [22], decreased astrocytic deal with those specific stressors. function leading to increased synaptic glutamate [23], re- duced trophic support to the neuron [24], microglial ac- Could toxicants in the locus ceruleus trigger MND? tivation and inflammation [25], and oligodendrocyte We were not able to detect heavy metals in cortical dysfunction causing impaired myelination [26]. Finally, it motor neurons, despite these neurons being affected in has been suggested that the LC could transfer toxicants ALS, and being shown to contain mercury after expos- from the circulation to cortical motor neurons [2], since ure to metallic mercury [2]. This could be because of a the LC makes direct contact with both capillaries and survivor effect, with the toxicant-containing cortical motor neurons. A further discussion on how toxic dam- motor neurons dying early and leaving only toxicant-free age to the LC could result in different neurological con- motor neurons intact. Secondly, toxicants within the LC ditions has recently been published [18]. may reduce noradrenaline output to the cortical motor No technique currently exists to reliably measure the neurons and secondarily damage cortical motor neurons, concentration of toxicants such as heavy metals within Figure 7 Potential interactions between toxicant-containing LC and motor neurons (see text for details). Toxicant uptake into the LC could reduce noradrenaline output, resulting in: (A) A dysfunctional blood-brain barrier, which becomes permeable to the same or other toxicants, or to inflammatory agents. (B) Decreased astrocytic function leading to increased synaptic glutamate. (C) Reduced trophic support to the neuron. (D) Microglial activation and inflammation. (E) Oligodendrocyte dysfunction causing impaired myelination. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 12 of 15 http://www.actaneurocomms.org/content/1/1/81 individual neurons, which raises the question of how to as- Phenotypic variation in familial and sporadic MND: do sess how much heavy metal is in LC neurons. The lack of toxicants play a part? obvious neuronal loss in the metal-containing LCs of our Heavy metal-containing neurons were found in this MND patients could lead to the presumption that the study in familial, and not only sporadic, MND. One pos- amount of heavy metal was insufficient to damage the neu- sibility for this is that FMND-associated mutations in- rons. However, it has previously been shown that mouse crease the neuronal uptake of toxicants, possibly by spinal motor neurons that contain a non-toxic dose of inor- altering the permeability of the blood–brain barrier as ganic mercury suffer axonal shrinkage, without any loss of has been suggested for SOD1 [34], TARDBP and ANG numbers of motor neuron cell bodies [9]. This indicates mutations [35]. Other FMND mutations could damage that a low dose of a heavy metal can cause neuronal dam- mechanisms that protect neurons from toxicants, as has age without cell body loss, and by extrapolation suggests a been suggested for a number of MND genetic variants loss of noradrenaline from LC terminals could occur with- [36]. out the loss of toxicant-containing LC cell bodies. Of inter- The presence of toxicants within LC and motor neu- est, one of the few quantitative studies of the LC in MND rons could also explain the intra-familial variations in has shown neuronal shrinkage of LC cell bodies without age of disease onset and phenotype that is seen in cell loss [27], though no unbiased quantitative studies of FMND, as well as the inter-patient variations commonly cell numbers in the LC of MND patients have been re- found in sporadic MND. For example, we found that the ported. Even a moderate level of LC neuronal damage heavy metal content varied between hypoglossal and could have a deleterious effect on spinal motor neurons, spinal motor neurons, a possible reason why bulbar since the long LC axons extending down to the spinal cord symptoms can appear early, late or not at all in MND. would be particularly susceptible to toxic disturbances in their parent cell bodies [28]. Heavy metal staining in non-MND disease controls AMG can detect only a few heavy metals, but environ- With the recent finding of LC damage in multiple scler- mental exposures usually involve different types of toxi- osis [37] it is of interest that the LC of one multiple cants, often simultaneously. For example, cigarette smoke, sclerosis patient contained heavy metals. The heavy a possible risk factor for MND [29], contains 4,800 identi- metal staining within the LC of one individual with alco- fied compounds, including metal toxicants [30]. The heavy holism accords with the finding by some workers that metals identified in this study may not therefore be a LC neurons are damaged by alcohol excess [38]. cause of neurotoxicity, but may merely be a marker that indicates how readily a number of toxicants are entering Identification and quantification of heavy metals in neurons LC and motor neurons. Ideally, AMG should be followed up with some elemental Although we have suggested here that heavy metals in method to confirm the identity of the heavy metal in the the LC could play a part in the pathogenesis of MND, tissue. This has been achievable in humans exposed to an alternative explanation for the presence of heavy large amounts mercury or bismuth, where enough of the metals in the LC is that metals enter the LC after the metal was present in the tissues for quantitative methods start of the disease. Stress-induced uptake of toxicants to be of use [12]. However, AMG, being an amplification could take place after the onset of MND, because having technique, is more sensitive to the presence of toxicant a disease such as MND is likely to be highly stressful. metals than other currently available techniques of metal The finding of heavy metals in the LC could therefore detection (such as neutron activation analysis, proton- be a result of having the disease, rather than being a induced X-ray emission, atomic absorption spectropho- trigger for the disease. tometry, and electron emission X-ray spectrophotometry), though the lower amount of heavy metal that can be de- Heavy metal staining in extraocular muscle neurons tected with AMG has not been ascertained [10]. Unfortu- Extraocular muscles are affected late or not at all in MND nately, microprobe elemental analyses performed on [31]. Extraocular muscle neurons contained the least formalin-fixed paraffin sections are considered to be unre- heavy metal staining of all the motor neurons in the liable [39]. In our study, only a small proportion of the present study, raising the possibility that one reason they total number of cells in the pons, brain stem and spinal are spared is because they take up smaller toxicant loads. cord had AMG staining, which suggests only an in situ This may be because their neuromuscular junctions are microprobe technique, carried out on frozen sections, different to those of other muscles [32]. Curiously, 2 out would have a chance of detecting toxicants in the LC. of 3 Parkinson’s disease patients had heavy metals in their Motor neuron loss in most MND cases was too severe to extraocular muscle neurons; whether this is related to the be able to identify toxicants using elemental analysis. oculomotor problems associated with Parkinson’s disease AMG has been used extensively to study the distribu- [33] would require further study. tion of metal toxicants in experimental animals, used Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 13 of 15 http://www.actaneurocomms.org/content/1/1/81 occasionally in humans with known heavy metal exposure available was limited, since spinal cord tissue was usu- [2,12,40], but used only rarely in humans with no known ally removed at post mortem from controls only when toxicant exposure [11]. Humans differ from most non- clinically indicated, and LC-containing blocks were not primate animals in having extensive neuromelanin pigmen- available from all potential controls. (2) Only one (vari- tation in catecholaminergic neurons, as well as having able) level of the LC was available for examination, so lipofuscin in many aging neurons, including motor neu- we were unable to determine if differences in heavy rons [41]. It is therefore important to ensure that the metal content were present in rostral, middle and caudal AMG staining seen in human neurons is related to heavy parts of the LC; of note, cell loss in the LC in Alzheimer's metal staining, and not to some unknown reaction within and Parkinson's diseases has been found to vary along the pigmented neurons. The fact that no heavy metal staining length of the LC [45]. (3) We could not reliably detect was seen in any neuromelanin-containing neurons of the the subceruleus, which in primates is thought to inner- substantia nigra, or in other neurons containing high vate spinal motor neurons [46]. (4) No histories of toxi- levels of lipofuscin, indicates that these pigments them- cant exposure were available, so we do not know if any selves areunlikelytobethe source of theAMG staining. individuals had exposure to higher than normal levels of The heavy metals in this study localised preferentially mercury (e.g., living near an incinerator or a coal-burning to the neuromelanin of LC neurons, and to the lipofus- power station) or had been taking bismuth-containing cin of motor neurons, probably because these pigments medications. (5) No psychological, psychiatric, or stressful can bind metals [41]. Neuromelanin in the substantia life events histories were available, so we were unable to nigra of neurologically-normal individuals has an affin- determine levels of stress before the onset of MND. This ity for a number of physiologic and exogenous metal is of potential importance, since it has been suggested that ions, including mercury [42]. It has been suggested that stress, by activating the LC, could increase the uptake of the neuromelanin of the substantia nigra could play dif- toxicants into LC neurons [2]. (6) As noted above, we ferent roles in Parkinson’s disease, firstly as a protect- were unable to identify the type of heavy metal using par- ant by scavenging toxic substances such as exogenous affin sections, though this is likely to be inorganic mercury metals and pesticides, and later as a destructive agent since human exposure to bismuth is limited. when cell death releases the neuromelanin [43]. Neuro- melanin granules are probably not membrane-bound, Future studies and toxicants in unbound neuromelanin would be free The finding of potential disease-causing toxicants in a to interact with cellular constituents [41]. CNS region that is not morphologically damaged by Quantitation of the metal content of neuromelanin MND has implications for further neurotoxicological in- isolated from the substantia nigra of neurologically- vestigations into this disease. Attempts to locate toxicants normal individuals shows how neuromelanin can se- in MND tissues have generally been unsuccessful, prob- quester toxic metals that arise from environmental ably because toxicant-containing motor neurons have exposure, with many-fold accumulations of mercury largely disappeared by the time of death, and bulk ana- (1:96) and lead (1:1408) compared to surrounding tis- lysis of tissue is unlikely to detect toxicants affecting a sue (no corresponding data are available for LC neuro- small percentage of cells. An intact LC, however, is com- melanin) [44]. Of note, however, was the absence of pact and easily located, and would be an ideal candi- stainable heavy metals in any substantia nigra neurons date region for newer microanalysis methods that can in our study, despite the probability of age-related detect a range of toxicants [47]. heavy metal sequestration by neuromelanin in these The heavy metals within LC neurons in the present neurons. This suggests that the AMG staining we did study did not appear to cause structural damage to the see in the LC, especially in densely-stained neurons, neurons, and only minor changes in LC neuronal size represents a concentration of heavy metal that could have been reported in MND [27,48]. There are however be physiologically active. Indirect evidence of the po- a number of neurodegenerative and psychiatric disorders tential toxicity of the level of AMG staining seen is also where structural damage to the LC with associated nor- given by the findings from a man who injected himself adrenaline deficits have been described. Chief among with a large amount of metallic mercury [2], after these are Parkinson’s disease, Alzheimer’s disease, major which similar AMG staining of many LC neurons was depression and bipolar disorder [18,49,50]. In Alzheimer's seen, comparable in particular to the densely-stained disease it has been suggested that the LC is the first region neurons in the present study. of the brain to be involved by the disease, and at a very early age [51]. Environmental toxicants have been associ- Study limitations ated with all these disorders, and a search for toxicants Our study has a number of limitations: (1) The number within the LC of these patients could yield interesting of controls who had both LC and spinal cord tissue results. Pamphlett and Kum Jew Acta Neuropathologica Communications 2013, 1:81 Page 14 of 15 http://www.actaneurocomms.org/content/1/1/81 Conclusion 8. Danscher G, Stoltenberg M, Kemp K, Pamphlett R: Bismuth autometallography: protocol, specificity, and differentiation. J Histochem In conclusion, we have shown that the uptake of heavy Cytochem 2000, 48:1503–1510. metals by the human LC is a fairly common occurrence, 9. 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Submit your next manuscript to BioMed Central and take full advantage of: doi:10.1186/2051-5960-1-81 Cite this article as: Pamphlett and Kum Jew: Heavy metals in locus ceruleus and motor neurons in motor neuron disease. Acta • Convenient online submission Neuropathologica Communications 2013 1:81. • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Published: Dec 12, 2013

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