BACKGROUND
wilson’s disease  is a disorder of copper  disposition of autosomal recessive inheritance
<cit> .
as a consequence, excessive cu accumulates in tissues and causes organ damage and malfunction which is responsible for the incidence of a wide spectrum of hepatic and neuropsychiatric symptoms.
the affected gene, i.e.
atp7b is located on the long arm of human chromosome  <dig> and encodes a cu-transporting p-type adenosine triphosphatase, atpase7b, which is beneath atpase7a the second known cu efflux pump.
in contrast to almost all other nutrients, cu passively enters the cell via channels such as ctr <dig> and excess cu is actively sequestered.
in polarized epithelial cell types forming inner surfaces such as gut enterocytes, choroid plexus ependymocytes and also capillary endothelial cells atpase7a is localized to the basolateral membrane and atpase7b is localized to the apical membrane
<cit> .
while those contain both isoforms, hepatocytes almost exclusively contain atpase7b.
consequently, atpase7a is responsible for absorption of cu from the intestine into the blood or from the capillary lumen into the tissue parenchyma while atpase7b is responsible for sequestration of cu into the bile, into the cerebrospinal fluid or back from an organ parenchyma into the circulation.
cu efflux may sequentially occur by separate sequestration of cu and coeruloplasmin apoprotein into trans-golgi vesicles, followed by formation of the tight complex there and subsequent exocytosis.
although cu is an essential cofactor for a number of enzymes and required for numerous cellular processes, cu overload is highly toxic resulting in oxidative damage and inflammation.
within the brain those wd patients with neurological symptoms showed lesions or atrophia in the basal ganglia and other regions
<cit> .
to our knowledge no data is available on cu concentrations in regions of post mortem human wd brain.
the initial neurological symptoms of cu overload may be very subtle, such as mild tremor, as well as speech and writing problems
<cit> .
at the molecular level these alterations are mainly caused by metal dependent production of dangerous radical oxygen species and the ability of cu to affect the secretion of molecules involved in the protection of neurons against oxidative stress
<cit> .
there are several rodent models of cu overload available that generate defects that closely resemble those found in patients suffering from wd.
historically, the first model described were long-evans cinnamon rats that carry a transcriptional deficient atp7b mutant in which a deletion removes over  <dig> bp of the coding region at the 3′ end that encodes the crucial highly conserved atp binding domain
.
similarly, the toxic milk mutation in mice in which a single base difference within the atp7b gene causes hepatic accumulation of cu that can be 100-fold higher than that observed in normal adults is also a well-accepted animal model for wd
.
the respective phenotype of these mice resembles in part that seen in patients with wd
<cit> .
a recent report has shown that the toxic milk mice accumulate cu in different regions of the brain resulting in an approximately twofold higher mean brain cu concentration at an age of 12 months than that observed in age-matched control mice
<cit> .
in addition, the same study reported that the cerebral zinc  concentration in 10 months animals was slightly higher than in control mice and that further iron  accumulation was found in the hippocampus of older animals.
in this model, elevated concentrations of cu were mainly found in the striatum, hippocampus and cerebellum but not in the cerebral cortex
<cit> .
these alterations were accompanied by a mild impairment in the rotarod and cylinder tests and further by a lack of acquisition of spatial memory in the morris water maze
<cit> .
in this regard, the phenotype of toxic milk mice resembles the clinical symptoms of patients suffering from wd.
however, this model has some limitations.
first, brain lesions in 12-month-old mice are not as advanced as in patients with the neurological form of wd
<cit> .
second, pups are born cu-deficient and the milk produced by mutant mothers contains too low amounts of cu resulting in early death, a characteristic that is not seen in patients affected by wd
<cit> .
moreover, morphologic and chemical studies of hepatic cu toxicosis indicated striking differences in morphologic integrity between regenerative nodules and the intervening parenchyma with profound changes in mitochondria, endoplasmic reticula, and nuclei as well as the accumulation of microvesicular lipid droplets in injured hepatocytes, again features that are not seen in patients with wd
<cit> .
in addition, despite a significant elevation of cu in the brain of the cu-loaded toxic milk mice no behavioral changes were observed in other studies
<cit>  raising some doubts about whether this model is appropriately mimicking wd
<cit> .
atp7b deficient mice that were generated by directed genetic disruption display a gradual accumulation of hepatic cu that increases to a level 60-fold greater than normal by 5 months of age and develop cirrhosis of the liver, while the progeny of homozygous mutant females are initially characterized by neurological abnormalities and growth retardation that is specific to cu deficiency
<cit> .
in addition, it is known that these mice show an increase in cu and fe concentration in the brain
<cit> .
interestingly, these mice have further elevated hepatic and cerebral superoxide dismutase activities indicating oxidative stress in respective tissues that is combined with a severe dysfunction of mitochondrial energy production
<cit> .
using quantitative positron emission tomography in atp7b deficient mice, it was demonstrated that injection of 64cucl <dig> intravenously resulted in increased accumulation and markedly reduced clearance of cu from the liver, while 64cu radioactivity at all times was lower in the brain, kidneys, lungs, and heart compared to wild type  animals suggesting that the reduced 64cu radioactivity in the atp7b null mice in extrahepatic tissues may be caused by hepatic trapping of cu
<cit> .
likewise, the oral administration of radioactive cucl <dig> was not able to induce higher concentrations of cu within brains of mice that lack functional atp7b protein
<cit> .
these imaging findings demonstrate that cu homeostasis and distribution in different organs is differentially regulated.
laser ablation inductively coupled plasma mass spectrometry  is a method capable of simultaneously detecting numerous metals and non-metals in one sample at concentrations as low  <dig> –1 μg g− <dig> for mn, fe, cu, zn and  <dig> – <dig>  μg g− <dig> for lanthanides
<cit> .
in the present study we performed la-icp-ms measurements in brains isolated from atp7b deficient mice and respective controls.
furthermore, gene expression and protein levels of a panel of inflammatory and blood vessel proliferation markers were studied.
RESULTS
metal accumulation in the brain and liver is a well known consequence of wd.
studies using magnetic resonance imaging have shown that patients with wd show bilateral t <dig> hyperintensity involving thalami, cerebral white matter, basal ganglia, cerebellum and brainstem
<cit> .
cu loading studies in the toxic milk mouse model  have shown that there are some chronological changes in tissue cu loading that also affect the bio-distribution of other metals
<cit> .
a recent publication analyzing this model has further shown that the amount of cu overload was unequally distributed in different brain regions
<cit> .
while the concentration was increased in the striatum, hippocampus and cerebellum, the concentration of cu was unaltered in the cerebral cortex
<cit> .
however, all these former studies used atomic absorption spectroscopy to measure metal concentrations in total extracts of the brain or its different areas and a more precise study of cu distribution in brain and its impact on the cerebral concentration of other metals or metalloids is presently not available in any experimental models of wd.
recently, we have shown that metal bio-imaging via la-icp-ms is a novel, highly sensitive innovative diagnostic tool that allows simultaneous measurement of over fifty different metal concentrations in liver tissue with a high sensitivity, spatial resolution, specificity, and quantification ability
<cit> .
we here used this methodology to get more insight into the cerebral distribution of various metals during progression of wd.
to do so, we comparatively imaged brain sections of wt and atp7b deficient mice at different ages by la-icp-ms. the horizontal sections analyzed contained cortex, corpus callosum, hippocampus, colliculus superior, cerebellum and all four ventricules in all cases, colliculus inferior in most cases, striatum, thalamus and brain stem structures in some cases .figure  <dig>
schematic overview of the murine brain and position of the sectional plane used for preparing cryosections.
in the scheme, the different regions and the architecture of the mouse brain are depicted.
the red lines mark the position for sectioning and the region from which horizontal cryosections were prepared.
a photomicrograph of a representative cryosection used for imaging is shown.
since numerous findings have already uniformly suggested that neuropathological alterations such as found in alzheimer’s disease, down syndrome, parkinson’s disease, multiple sclerosis, and many other neurodegenerative disorders are characterized or even causatively driven by altered expression of il-1β
, we first comparatively tested the mrna expression of this cytokine in wt and atp7b deficient mice .
in contrast to all these previous reports, we found that the il-1β mrna quantities were only slightly but not significantly elevated in wd vs. controls.
tnf-α mrna expression, in turn, was significantly increased more than two-fold in wd.
tnf-α is associated with damage of myelin, oligodendrocytes and astrocytes
<cit>  and may possibly reflect ongoing neurotoxicity
<cit> .
in line with this hypothesis, we found increased expression of the nlrp <dig> and asc inflammasome components in the brains of wd mice.
those components have important roles in the pathology of microglia-specific formation of neurodegenerative disorders and further regulate cns inflammation and de-myelination during cerebral insult
<cit> .
this finding again demonstrates that affected mice have exacerbating cns inflammatory activity within their brains.
in addition, both, the tissue inhibitor of metalloproteinase- <dig>  and the matrix metalloproteinase  <dig>  that represent biomarkers for the presence or extent of brain injury and are necessary for the clearance of amyloid
<cit>  were expressed at higher levels in atp7b deficient brains.
since mmps promote blood–brain barrier leakage, it is possible that the elevated expression of mmp- <dig> may already indicate existing brain cell dysfunction and cerebral cell death
<cit> .
we next performed western blot analysis and found that the expression of α-smooth muscle actin  in protein extracts prepared from the brains of atp7b knockout mice  was significantly higher .
in normal brain tissue, α-sma is generally undetectable and it was previously supposed that α-sma should be considered as a marker for smooth muscle cell differentiation, which in the brain becomes detectable in areas with microvascular proliferation
<cit> .
again, these findings indicate that the atp7b nulls already show signs of cerebral dysfunction.
the increased activation of inflammatory pathways within the brains that we have already observed at the transcriptional level was also noticeable in the elevated concentration of the lipocalin  <dig>  in older animals that lack atp7b.
we have recently demonstrated that the upregulation of lcn <dig> is a reliable indicator of organ damage that is significantly correlated to inflammation
<cit> .
altogether, our gene and protein expression data indicate that the atp7b deficient mice have some cerebral alterations that are characteristic for wd in humans.
la-icp-ms simultaneously yielded images of the  <dig> preselected m/z.
amongst these elements we first inspected the distribution of sodium and phosphorus within the brains of wt and atp7b deficient mice in cryosections with a thickness of 30 μm
<cit>  resulting in a maintained architecture of these elements.
in the unchanged physiological situation highest concentrations of phosphorus  were found in the granular layer of the cerebellum forming the shape of the arbor vitae, the granular layer of the olfactory bulb, in the intrastriatal and intracollicular fiber bundles and corpus callosum .
sodium in the well known manner showed the distribution of water with higher concentrations in grey matter 800 μg g− <dig> compared to white matter 600 μg g− <dig>  inversely, 13c ion intensities reflected the distribution of dry organic matter congruent with the distribution of the fatty white matter.
next manganese , fe, cu and zn were assessed quantitatively in the regions of interest exemplarily depicted in figure
<dig>  average concentrations read out from large regions are given in table
<dig> and those of small hippocampal regions in table
<dig>  while fe and mn were unchanged and of perfectly preserved distributional architecture a tremendous increase of cu was already obvious from visual inspection .
at a second glance the decrease of cu in the periventricular regions was obvious, especially in the fourth ventricule where a lumen was systematically discernable in wd animals but not in controls.
otherwise the physiological distribution pattern of cu was maintained.
this metal is the element presenting the highest range of physiological concentrations.
in wt controls cu ranged from a minimum in callosal white matter of  <dig>  μg g− <dig> to 67 μg g− <dig> in the fourth ventricule.
in the other grey matter components cu occurs in quite low concentrations such as  <dig>  μg g− <dig> in the cortex and  <dig>  μg g− <dig> in the colliculus superior.
somewhat higher cu was observed in the stratum moleculare of the fascia dentata , the preasubiculum  and in the glomerular layer of the olfactory bulb.
within the isocortex cu was slightly higher in the layers  <dig>   <dig> and  <dig>  for validation additional  <dig> wd mice vs.  <dig> age-matched controls were studied by the generation of quantitative images only but not by region of interest based analysis.
here especially high quality mn images were obtained and all above mentioned findings could be reproduced .figure  <dig>
bioimaging of sodium and phosphate in the murine brain.
thin brain section of wt  and atp7b deficient mice  were prepared by cryo-cutting and analyzed for na and p content by la-icp-ms. in these measurements, the metal intensities were normalized to the average 13c ion intensity in respective samples.
the photomicrographs of the analyzed cryo-sections are depicted for orientation.
regions of interest.
exemplarily different regions of the murine brain in which metal content was measured by la-icp-ms and quantified  are depicted.
average concentration of fe, cu, zn and mn in large regions of the brain
concentrations of fe, cu, zn, mn  measured by imaging la-icp-ms in regions of interest in the brain of  <dig> controls, and  <dig> wd mice, both on a svj <dig> background.
of each animal, one section was measured whereby each hemisphere was measured in an independent run with separate calibration standard.
sd and heteroskedastic t-tests were calculated over the  <dig> and  <dig> individual bi-hemispheric averages.
*white matter borders were delineated to minimize spillover from neighbour regions.
**indicated, if > +10% or < −10% and p < .
<dig>
average concentration of fe, cu, zn and mn in small hippocampal regions of the brain
concentrations of fe, cu, zn, mn  measured by la-icp-msi in subregions of the hippocampal formation.
layers are given in the order from outside to inside.
age-dependent cerebral accumulation of copper in
atp7b
−/−
mice as demonstrated by la-icp-ms. 30 μm thick cryosections from wt  and atp7b
−/− mice  at age between  <dig> and 24 months were subjected to la-icp-ms imaging.
the content of carbon that served as reference in this analysis is given in %, while the total concentrations of cu, zn, and fe are given in μg g− <dig> tissue.
high resolution imaging of copper within the brain of age-matched wild type and
atp7b
deficient mice.
brain sections of a wt  and atp7b deficient mice  were prepared from age-matched  old animals and subjected to la-icp-ms imaging to estimate the content of copper.
carbon served as an internal standard in this analysis.
quantitative analysis confirmed an almost proportional increase of cu by 50%-130% in the brain parenchyma of atp7b deficient animals without further obvious systematic between more or less cu rich regions.
this finding reached significances of p =  <dig>  fulfilling even the most conservative bonferroni criteria.
in the periventricular regions in turn cu was decreased by factors of  <dig>  in and around ventricules i-iii and of  <dig>  around ventricule iv.
interestingly, also the gradient of cu concentrations increasing downstream from lateral over third to fourth ventricule was relieved.
in the choroid plexus the cerebrospinal fluid  is produced essentially by ultrafiltration through fenestrated capillaries.
a part of the initially co-filtrated cu is immediately re-absorbed through ependymocytes which express high amounts of atpase7a at their basolateral membrane.
ependymocytes of the ventricular walls sequester cu from brain parenchyma to the csf via atpase7b localized at their apical portion.
it is fully compatible with the view of periventricular regions as cu efflux compartments that a lack of function of atpase7b leads to a decrease of cu there.
zn was increased by up to 40% especially in regions of high cu such as the stratum moleculare of the fascia dentata or cu rich areas of the cerebellum but not in typical zn accumulator regions, such as the stratum multiforme of the fascia dentata and the ca <dig> segment of the pyramidal layer of the cornu ammonis, a side effect due to zn being to some degree a substrate of cu-atpases.
on the other hand a significant increase of zn was observed in the zn rich ectorhinal cortex  and anterior cingulate cortex .
to avoid potential misinterpretation of results that may occur from animal-to-animal variations, we further performed confirmatory measurements in additional  <dig> wd and  <dig> wt controls that were at age of 19– <dig> months.
the visual inspection of the images that were generated by the in house written excel algorithm for visualization of metal concentrations  results in similar images that confirmed our measurements .
therefore, these measurements were not further submitted to region of interest based analysis.
when it comes to variation of metal concentrations with age, the number of sections and animals submitted to quantitative data analysis here is too small for final conclusions.
however, it can already be stated that in this sample the full extent of altered cu disposition was already reached at 11 months and concentrations of cu, zn and mn rather slightly fall with age while fe was constant in wd.
the more interesting period from  <dig> to 11 months was not covered by this sample.
here, in a previous study in which selected elements were quantified by atomic absorption spectroscopy
<cit>  some of us reported a progressive increase of cu and zn.
the animals used were aged  <dig>   <dig>  and 47 weeks .
fe had reached its full level already in 6 week old mice
<cit> .
based on our findings, we could confirm that the homozygous atp7b deficient mice used in our study accumulate cerebral cu.
a previous study that has reported the establishment of the respective animal model has already demonstrated by use of polarized atomic absorption spectrophotometry of dried tissue samples that the levels of cu in brain of homozygous mutant animals increased slightly throughout adult life from  <dig>  ±  <dig>  to 36 ± 4 μg g− <dig> dry weight
<cit> .
in human wd
<cit> , the functionality controlled by regions such as the basal ganglia, namely striatum and pallidum is tremendously affected potentially indicating that the observed alterations might be a direct consequence of cu deposits there
<cit> .
in line with this, the olfactory bulb that is located in the forebrain contains specialized sensory cells and interneurons responsible for accurate transmission of smell information
<cit> .
in wd patients with neurological symptoms a significant irreversible olfactory dysfunction that correlates with cu content is documented
<cit> .
it was recently demonstrated that increased brain cu in the toxic milk mice model correlated well with the expression of inflammatory markers and behavioral changes
<cit> .
immunohistochemical determination of atp7b protein in brain tissue of rats has shown that this cu-transporting p-type atpase was highly expressed in neuronal cells of the hippocampal formation, olfactory bulbs, cerebellum, cerebral cortex and nuclei in the brainstem
<cit> .
the finding that cu is also enriched in the cerebral cortex when atp7b is absent confirms the hypothesized essential role of this gene in the local export of cu from cells
<cit> .
somewhat in contrast to our findings is the observation that intravenous injection of 64cucl <dig> via the tail vein resulted in consistently lower cu radioactivity at all times in the brain of atp7b knockout mice as compared to control mice when analyzed by quantitative positron emission tomography
<cit> .
in this context, it has to be well discriminated that this pet study measured the uptake and turnover but not the total inventory of cu as it was the case in our study.
bio-distribution studies with application of radio-cu at lifetime, victimization after hours and few days and autoradiography of brain sections clearly localized the fastest and highest uptake of cu to the periventricular regions
<cit> .
thus lower uptake of cu into periventricular regions in wd is perfectly in line with the finding of lower cu there in this study in wd mice.
another report that was conducted in the toxic milk mouse model on the c3h background has demonstrated that the cu accumulation was mainly found in striatum, hippocampus and cerebellum, but unaltered in cerebral cortex
<cit> .
the latter finding may be explained by a systematic contamination/mix-up of dissected cortical material with periventricular material  and thus wrongly high cortical cu levels in controls.
these mice showed motor and cognitive disturbances most likely because of the cu deposition and inflammatory response observed in the striatum.
similarly, accumulation of fe in the brain of aged lec rats  was reported previously with highest levels in the striatum and the substantia nigra
<cit> .
there is no doubt that the la-icp-ms imaging technology used in this study is transferable to other experimental models, organs, species, and various metal overload diseases.
we here used mouse that lacked the plasma membrane cu-transport protein atp7b.
in future it will be interesting for us to visualize the metal content in supplementary rodent models of wd including the toxic milk mouse model or the long-evans cinnamon rats by la-icp-ms. in addition, recent reports have provided strong evidence that cu metabolism murri domain-containing protein  <dig> -deficiency in dogs lead to hepatic cu accumulation with a wd-like phenotype
<cit> .
also these models in conjunction with la-icp-ms will help to increase the knowledge in the pathogenesis of neurodegenerative disorders that occur during the course of metal accumulation.
CONCLUSIONS
our study evidenced an about 2-fold stable increase of cu throughout brain parenchyma in atpase7b null mice more or less proportional across the regions assessed maintaining the characteristic distribution pattern while in periventricular regions cu was decreased by down to a factor of  <dig> .
as cu was increased proportionally throughout all cerebral regions the regionally and basal ganglia characteristic symptoms of wd may rather result from differential regional susceptibility to cu than from differential affection of cu-efflux.
zn was increased by 10%-40%.
it is now tempting to assess horizontal sections at a deeper level covering the candidate structures pallidum, striatum and brain stem nuclei and completing cohort by animals of younger ages.
animal models deficient in cu transporters and la-icp-ms imaging are a setting perfectly suited to study therapeutic interventions for normalizing cu disposition and also studying their side effects on other metals, namely zn.
in a wider perspective, this study demonstrates the superiority of microlocal analytical techniques over the study of homogenates especially if regions or layers of highly differential analyte concentrations are closely adjacent which cannot be dissected reliably from each other.
la-icp-ms imaging is an important novel method that will be helpful in the setting of experimental and clinical research.
this method will be easily transferable to other models, organs and diseases and will have a strong influence on basic research and diagnostics in the near future.

 wilson’s disease is a disorder of copper disposition of autosomal recessive inheritance. as a consequence, excessive cu accumulates in tissues and causes organ damage and malfunction which is responsible for the incidence of a wide spectrum of hepatic and neuropsychiatric symptoms. the affected gene, i.e. atp7b is located on the long arm of human chromosome and encodes a cu-transporting p-type adenosine triphosphatase, atpase7b, which is beneath atpase7a the second known cu efflux pump. in contrast to almost all other nutrients, cu passively enters the cell via channels such as ctr and excess cu is actively sequestered. in polarized epithelial cell types forming inner surfaces such as gut enterocytes, choroid plexus ependymocytes and also capillary endothelial cells atpase7a is localized to the basolateral membrane and atpase7b is localized to the apical membrane. while those contain both isoforms, hepatocytes almost exclusively contain atpase7b. consequently, atpase7a is responsible for absorption of cu from the intestine into the blood or from the capillary lumen into the tissue parenchyma while atpase7b is responsible for sequestration of cu into the bile, into the cerebrospinal fluid or back from an organ parenchyma into the circulation. cu efflux may sequentially occur by separate sequestration of cu and coeruloplasmin apoprotein into trans-golgi vesicles, followed by formation of the tight complex there and subsequent exocytosis. although cu is an essential cofactor for a number of enzymes and required for numerous cellular processes, cu overload is highly toxic resulting in oxidative damage and inflammation. within the brain those wd patients with neurological symptoms showed lesions or atrophia in the basal ganglia and other regions. to our knowledge no data is available on cu concentrations in regions of post mortem human wd brain. the initial neurological symptoms of cu overload may be very subtle, such as mild tremor, as well as speech and writing problems. at the molecular level these alterations are mainly caused by metal dependent production of dangerous radical oxygen species and the ability of cu to affect the secretion of molecules involved in the protection of neurons against oxidative stress. there are several rodent models of cu overload available that generate defects that closely resemble those found in patients suffering from wd. historically, the first model described were long-evans cinnamon rats that carry a transcriptional deficient atp7b mutant in which a deletion removes over bp of the coding region at the 3′ end that encodes the crucial highly conserved atp binding domain. similarly, the toxic milk mutation in mice in which a single base difference within the atp7b gene causes hepatic accumulation of cu that can be 100-fold higher than that observed in normal adults is also a well-accepted animal model for wd. the respective phenotype of these mice resembles in part that seen in patients with wd. a recent report has shown that the toxic milk mice accumulate cu in different regions of the brain resulting in an approximately twofold higher mean brain cu concentration at an age of 12 months than that observed in age-matched control mice. in addition, the same study reported that the cerebral zinc concentration in 10 months animals was slightly higher than in control mice and that further iron accumulation was found in the hippocampus of older animals. in this model, elevated concentrations of cu were mainly found in the striatum, hippocampus and cerebellum but not in the cerebral cortex. these alterations were accompanied by a mild impairment in the rotarod and cylinder tests and further by a lack of acquisition of spatial memory in the morris water maze. in this regard, the phenotype of toxic milk mice resembles the clinical symptoms of patients suffering from wd. however, this model has some limitations. first, brain lesions in 12-month-old mice are not as advanced as in patients with the neurological form of wd. second, pups are born cu-deficient and the milk produced by mutant mothers contains too low amounts of cu resulting in early death, a characteristic that is not seen in patients affected by wd. moreover, morphologic and chemical studies of hepatic cu toxicosis indicated striking differences in morphologic integrity between regenerative nodules and the intervening parenchyma with profound changes in mitochondria, endoplasmic reticula, and nuclei as well as the accumulation of microvesicular lipid droplets in injured hepatocytes, again features that are not seen in patients with wd. in addition, despite a significant elevation of cu in the brain of the cu-loaded toxic milk mice no behavioral changes were observed in other studies raising some doubts about whether this model is appropriately mimicking wd. atp7b deficient mice that were generated by directed genetic disruption display a gradual accumulation of hepatic cu that increases to a level 60-fold greater than normal by 5 months of age and develop cirrhosis of the liver, while the progeny of homozygous mutant females are initially characterized by neurological abnormalities and growth retardation that is specific to cu deficiency. in addition, it is known that these mice show an increase in cu and fe concentration in the brain. interestingly, these mice have further elevated hepatic and cerebral superoxide dismutase activities indicating oxidative stress in respective tissues that is combined with a severe dysfunction of mitochondrial energy production. using quantitative positron emission tomography in atp7b deficient mice, it was demonstrated that injection of 64cucl intravenously resulted in increased accumulation and markedly reduced clearance of cu from the liver, while 64cu radioactivity at all times was lower in the brain, kidneys, lungs, and heart compared to wild type animals suggesting that the reduced 64cu radioactivity in the atp7b null mice in extrahepatic tissues may be caused by hepatic trapping of cu. likewise, the oral administration of radioactive cucl was not able to induce higher concentrations of cu within brains of mice that lack functional atp7b protein. these imaging findings demonstrate that cu homeostasis and distribution in different organs is differentially regulated. laser ablation inductively coupled plasma mass spectrometry is a method capable of simultaneously detecting numerous metals and non-metals in one sample at concentrations as low –1 μg g− for mn, fe, cu, zn and –  μg g− for lanthanides. in the present study we performed la-icp-ms measurements in brains isolated from atp7b deficient mice and respective controls. furthermore, gene expression and protein levels of a panel of inflammatory and blood vessel proliferation markers were studied.  metal accumulation in the brain and liver is a well known consequence of wd. studies using magnetic resonance imaging have shown that patients with wd show bilateral t hyperintensity involving thalami, cerebral white matter, basal ganglia, cerebellum and brainstem. cu loading studies in the toxic milk mouse model have shown that there are some chronological changes in tissue cu loading that also affect the bio-distribution of other metals. a recent publication analyzing this model has further shown that the amount of cu overload was unequally distributed in different brain regions. while the concentration was increased in the striatum, hippocampus and cerebellum, the concentration of cu was unaltered in the cerebral cortex. however, all these former studies used atomic absorption spectroscopy to measure metal concentrations in total extracts of the brain or its different areas and a more precise study of cu distribution in brain and its impact on the cerebral concentration of other metals or metalloids is presently not available in any experimental models of wd. recently, we have shown that metal bio-imaging via la-icp-ms is a novel, highly sensitive innovative diagnostic tool that allows simultaneous measurement of over fifty different metal concentrations in liver tissue with a high sensitivity, spatial resolution, specificity, and quantification ability. we here used this methodology to get more insight into the cerebral distribution of various metals during progression of wd. to do so, we comparatively imaged brain sections of wt and atp7b deficient mice at different ages by la-icp-ms. the horizontal sections analyzed contained cortex, corpus callosum, hippocampus, colliculus superior, cerebellum and all four ventricules in all cases, colliculus inferior in most cases, striatum, thalamus and brain stem structures in some cases.figure schematic overview of the murine brain and position of the sectional plane used for preparing cryosections. in the scheme, the different regions and the architecture of the mouse brain are depicted. the red lines mark the position for sectioning and the region from which horizontal cryosections were prepared. a photomicrograph of a representative cryosection used for imaging is shown. since numerous findings have already uniformly suggested that neuropathological alterations such as found in alzheimer’s disease, down syndrome, parkinson’s disease, multiple sclerosis, and many other neurodegenerative disorders are characterized or even causatively driven by altered expression of il-1β, we first comparatively tested the mrna expression of this cytokine in wt and atp7b deficient mice. in contrast to all these previous reports, we found that the il-1β mrna quantities were only slightly but not significantly elevated in wd vs. controls. tnf-α mrna expression, in turn, was significantly increased more than two-fold in wd. tnf-α is associated with damage of myelin, oligodendrocytes and astrocytes and may possibly reflect ongoing neurotoxicity. in line with this hypothesis, we found increased expression of the nlrp and asc inflammasome components in the brains of wd mice. those components have important roles in the pathology of microglia-specific formation of neurodegenerative disorders and further regulate cns inflammation and de-myelination during cerebral insult. this finding again demonstrates that affected mice have exacerbating cns inflammatory activity within their brains. in addition, both, the tissue inhibitor of metalloproteinase- and the matrix metalloproteinase that represent biomarkers for the presence or extent of brain injury and are necessary for the clearance of amyloid were expressed at higher levels in atp7b deficient brains. since mmps promote blood–brain barrier leakage, it is possible that the elevated expression of mmp- may already indicate existing brain cell dysfunction and cerebral cell death. we next performed western blot analysis and found that the expression of α-smooth muscle actin in protein extracts prepared from the brains of atp7b knockout mice was significantly higher. in normal brain tissue, α-sma is generally undetectable and it was previously supposed that α-sma should be considered as a marker for smooth muscle cell differentiation, which in the brain becomes detectable in areas with microvascular proliferation. again, these findings indicate that the atp7b nulls already show signs of cerebral dysfunction. the increased activation of inflammatory pathways within the brains that we have already observed at the transcriptional level was also noticeable in the elevated concentration of the lipocalin in older animals that lack atp7b. we have recently demonstrated that the upregulation of lcn is a reliable indicator of organ damage that is significantly correlated to inflammation. altogether, our gene and protein expression data indicate that the atp7b deficient mice have some cerebral alterations that are characteristic for wd in humans. la-icp-ms simultaneously yielded images of the preselected m/z. amongst these elements we first inspected the distribution of sodium and phosphorus within the brains of wt and atp7b deficient mice in cryosections with a thickness of 30 μm resulting in a maintained architecture of these elements. in the unchanged physiological situation highest concentrations of phosphorus were found in the granular layer of the cerebellum forming the shape of the arbor vitae, the granular layer of the olfactory bulb, in the intrastriatal and intracollicular fiber bundles and corpus callosum. sodium in the well known manner showed the distribution of water with higher concentrations in grey matter 800 μg g− compared to white matter 600 μg g− inversely, 13c ion intensities reflected the distribution of dry organic matter congruent with the distribution of the fatty white matter. next manganese, fe, cu and zn were assessed quantitatively in the regions of interest exemplarily depicted in figure  average concentrations read out from large regions are given in table  and those of small hippocampal regions in table  while fe and mn were unchanged and of perfectly preserved distributional architecture a tremendous increase of cu was already obvious from visual inspection. at a second glance the decrease of cu in the periventricular regions was obvious, especially in the fourth ventricule where a lumen was systematically discernable in wd animals but not in controls. otherwise the physiological distribution pattern of cu was maintained. this metal is the element presenting the highest range of physiological concentrations. in wt controls cu ranged from a minimum in callosal white matter of  μg g− to 67 μg g− in the fourth ventricule. in the other grey matter components cu occurs in quite low concentrations such as  μg g− in the cortex and  μg g− in the colliculus superior. somewhat higher cu was observed in the stratum moleculare of the fascia dentata, the preasubiculum and in the glomerular layer of the olfactory bulb. within the isocortex cu was slightly higher in the layers and for validation additional wd mice vs. age-matched controls were studied by the generation of quantitative images only but not by region of interest based analysis. here especially high quality mn images were obtained and all above mentioned findings could be reproduced.figure bioimaging of sodium and phosphate in the murine brain. thin brain section of wt and atp7b deficient mice were prepared by cryo-cutting and analyzed for na and p content by la-icp-ms. in these measurements, the metal intensities were normalized to the average 13c ion intensity in respective samples. the photomicrographs of the analyzed cryo-sections are depicted for orientation. regions of interest. exemplarily different regions of the murine brain in which metal content was measured by la-icp-ms and quantified are depicted. average concentration of fe, cu, zn and mn in large regions of the brain concentrations of fe, cu, zn, mn measured by imaging la-icp-ms in regions of interest in the brain of controls, and wd mice, both on a svj background. of each animal, one section was measured whereby each hemisphere was measured in an independent run with separate calibration standard. sd and heteroskedastic t-tests were calculated over the and individual bi-hemispheric averages. *white matter borders were delineated to minimize spillover from neighbour regions. **indicated, if > +10% or < −10% and p <. average concentration of fe, cu, zn and mn in small hippocampal regions of the brain concentrations of fe, cu, zn, mn measured by la-icp-msi in subregions of the hippocampal formation. layers are given in the order from outside to inside. age-dependent cerebral accumulation of copper in atp7b −/− mice as demonstrated by la-icp-ms. 30 μm thick cryosections from wt and atp7b −/− mice at age between and 24 months were subjected to la-icp-ms imaging. the content of carbon that served as reference in this analysis is given in %, while the total concentrations of cu, zn, and fe are given in μg g− tissue. high resolution imaging of copper within the brain of age-matched wild type and atp7b deficient mice. brain sections of a wt and atp7b deficient mice were prepared from age-matched old animals and subjected to la-icp-ms imaging to estimate the content of copper. carbon served as an internal standard in this analysis. quantitative analysis confirmed an almost proportional increase of cu by 50%-130% in the brain parenchyma of atp7b deficient animals without further obvious systematic between more or less cu rich regions. this finding reached significances of p =  fulfilling even the most conservative bonferroni criteria. in the periventricular regions in turn cu was decreased by factors of in and around ventricules i-iii and of around ventricule iv. interestingly, also the gradient of cu concentrations increasing downstream from lateral over third to fourth ventricule was relieved. in the choroid plexus the cerebrospinal fluid is produced essentially by ultrafiltration through fenestrated capillaries. a part of the initially co-filtrated cu is immediately re-absorbed through ependymocytes which express high amounts of atpase7a at their basolateral membrane. ependymocytes of the ventricular walls sequester cu from brain parenchyma to the csf via atpase7b localized at their apical portion. it is fully compatible with the view of periventricular regions as cu efflux compartments that a lack of function of atpase7b leads to a decrease of cu there. zn was increased by up to 40% especially in regions of high cu such as the stratum moleculare of the fascia dentata or cu rich areas of the cerebellum but not in typical zn accumulator regions, such as the stratum multiforme of the fascia dentata and the ca segment of the pyramidal layer of the cornu ammonis, a side effect due to zn being to some degree a substrate of cu-atpases. on the other hand a significant increase of zn was observed in the zn rich ectorhinal cortex and anterior cingulate cortex. to avoid potential misinterpretation of results that may occur from animal-to-animal variations, we further performed confirmatory measurements in additional wd and wt controls that were at age of 19– months. the visual inspection of the images that were generated by the in house written excel algorithm for visualization of metal concentrations results in similar images that confirmed our measurements. therefore, these measurements were not further submitted to region of interest based analysis. when it comes to variation of metal concentrations with age, the number of sections and animals submitted to quantitative data analysis here is too small for final conclusions. however, it can already be stated that in this sample the full extent of altered cu disposition was already reached at 11 months and concentrations of cu, zn and mn rather slightly fall with age while fe was constant in wd. the more interesting period from to 11 months was not covered by this sample. here, in a previous study in which selected elements were quantified by atomic absorption spectroscopy some of us reported a progressive increase of cu and zn. the animals used were aged and 47 weeks. fe had reached its full level already in 6 week old mice. based on our findings, we could confirm that the homozygous atp7b deficient mice used in our study accumulate cerebral cu. a previous study that has reported the establishment of the respective animal model has already demonstrated by use of polarized atomic absorption spectrophotometry of dried tissue samples that the levels of cu in brain of homozygous mutant animals increased slightly throughout adult life from  ±  to 36 ± 4 μg g− dry weight. in human wd, the functionality controlled by regions such as the basal ganglia, namely striatum and pallidum is tremendously affected potentially indicating that the observed alterations might be a direct consequence of cu deposits there. in line with this, the olfactory bulb that is located in the forebrain contains specialized sensory cells and interneurons responsible for accurate transmission of smell information. in wd patients with neurological symptoms a significant irreversible olfactory dysfunction that correlates with cu content is documented. it was recently demonstrated that increased brain cu in the toxic milk mice model correlated well with the expression of inflammatory markers and behavioral changes. immunohistochemical determination of atp7b protein in brain tissue of rats has shown that this cu-transporting p-type atpase was highly expressed in neuronal cells of the hippocampal formation, olfactory bulbs, cerebellum, cerebral cortex and nuclei in the brainstem. the finding that cu is also enriched in the cerebral cortex when atp7b is absent confirms the hypothesized essential role of this gene in the local export of cu from cells. somewhat in contrast to our findings is the observation that intravenous injection of 64cucl via the tail vein resulted in consistently lower cu radioactivity at all times in the brain of atp7b knockout mice as compared to control mice when analyzed by quantitative positron emission tomography. in this context, it has to be well discriminated that this pet study measured the uptake and turnover but not the total inventory of cu as it was the case in our study. bio-distribution studies with application of radio-cu at lifetime, victimization after hours and few days and autoradiography of brain sections clearly localized the fastest and highest uptake of cu to the periventricular regions. thus lower uptake of cu into periventricular regions in wd is perfectly in line with the finding of lower cu there in this study in wd mice. another report that was conducted in the toxic milk mouse model on the c3h background has demonstrated that the cu accumulation was mainly found in striatum, hippocampus and cerebellum, but unaltered in cerebral cortex. the latter finding may be explained by a systematic contamination/mix-up of dissected cortical material with periventricular material and thus wrongly high cortical cu levels in controls. these mice showed motor and cognitive disturbances most likely because of the cu deposition and inflammatory response observed in the striatum. similarly, accumulation of fe in the brain of aged lec rats was reported previously with highest levels in the striatum and the substantia nigra. there is no doubt that the la-icp-ms imaging technology used in this study is transferable to other experimental models, organs, species, and various metal overload diseases. we here used mouse that lacked the plasma membrane cu-transport protein atp7b. in future it will be interesting for us to visualize the metal content in supplementary rodent models of wd including the toxic milk mouse model or the long-evans cinnamon rats by la-icp-ms. in addition, recent reports have provided strong evidence that cu metabolism murri domain-containing protein -deficiency in dogs lead to hepatic cu accumulation with a wd-like phenotype. also these models in conjunction with la-icp-ms will help to increase the knowledge in the pathogenesis of neurodegenerative disorders that occur during the course of metal accumulation.  our study evidenced an about 2-fold stable increase of cu throughout brain parenchyma in atpase7b null mice more or less proportional across the regions assessed maintaining the characteristic distribution pattern while in periventricular regions cu was decreased by down to a factor of. as cu was increased proportionally throughout all cerebral regions the regionally and basal ganglia characteristic symptoms of wd may rather result from differential regional susceptibility to cu than from differential affection of cu-efflux. zn was increased by 10%-40%. it is now tempting to assess horizontal sections at a deeper level covering the candidate structures pallidum, striatum and brain stem nuclei and completing cohort by animals of younger ages. animal models deficient in cu transporters and la-icp-ms imaging are a setting perfectly suited to study therapeutic interventions for normalizing cu disposition and also studying their side effects on other metals, namely zn. in a wider perspective, this study demonstrates the superiority of microlocal analytical techniques over the study of homogenates especially if regions or layers of highly differential analyte concentrations are closely adjacent which cannot be dissected reliably from each other. la-icp-ms imaging is an important novel method that will be helpful in the setting of experimental and clinical research. this method will be easily transferable to other models, organs and diseases and will have a strong influence on basic research and diagnostics in the near future. 
in the periventricular regions known as structures of prominent cu content, cu was reduced in turn by a factor of this corroborates the view of the pvr as efflux compartments with active transport of cu into the cerebrospinal fluid. this study confirmed stable cerebral cu accumulation in parenchyma and discovered reduced cu in cerebrospinal fluid in atp7b null mice underlining the diagnostic value of micro-local analytical techniques. we could confirm cu accumulation in brain parenchyma by a factor of two in wd vs. controls that was already fully established at 11 months. otherwise the architecture of cu distribution was essentially maintained. we have applied laser ablation inductively coupled plasma mass spectrometry and performed comparative metal bio-imaging in brain sections of wild type and atp7b null mice in the age range of 11– months. messenger rna and protein expression of a panel of inflammatory markers were assessed using rt-pcr and western blots of brain homogenates. inflammatory markers tnf-α, timp- and the capillary proliferation marker α-sma were increased by a factor of 2– in wd. zinc was increased by up to 40% especially in regions of high cu but not in typical zn accumulator regions, a side effect due to the fact that zn is to some degree a substrate of cu-atpases. these show a gradual accumulation and concentration of cu in liver, kidneys, and brain.
 neuropsychiatric affection involving extrapyramidal symptoms is a frequent component of wilson’s disease. wd is caused by a genetic defect of the copper efflux pump atpase7b. mouse strains with natural or engineered transgenic defects of the atp7b gene have served as model of wd. these show a gradual accumulation and concentration of cu in liver, kidneys, and brain. however, still little is known about the regional distribution of cu inside the brain, its influence on other metals and subsequent pathophysiological mechanisms. we have applied laser ablation inductively coupled plasma mass spectrometry and performed comparative metal bio-imaging in brain sections of wild type and atp7b null mice in the age range of 11– months. messenger rna and protein expression of a panel of inflammatory markers were assessed using rt-pcr and western blots of brain homogenates.  we could confirm cu accumulation in brain parenchyma by a factor of two in wd vs. controls that was already fully established at 11 months. in the periventricular regions known as structures of prominent cu content, cu was reduced in turn by a factor of this corroborates the view of the pvr as efflux compartments with active transport of cu into the cerebrospinal fluid. furthermore, the gradient of cu increasing downstream the pvr was relieved. otherwise the architecture of cu distribution was essentially maintained. zinc was increased by up to 40% especially in regions of high cu but not in typical zn accumulator regions, a side effect due to the fact that zn is to some degree a substrate of cu-atpases. the concentrations of iron and manganese were constant throughout all regions assessed. inflammatory markers tnf-α, timp- and the capillary proliferation marker α-sma were increased by a factor of 2– in wd.  this study confirmed stable cerebral cu accumulation in parenchyma and discovered reduced cu in cerebrospinal fluid in atp7b null mice underlining the diagnostic value of micro-local analytical techniques. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords wilson’s diseasebio-imagingla-icp-mscopperatpase7bissue-copyright-statement© the author 2014 