Magnetic iron-oxide nanoparticles in the brain connected to alcohol-associated liver disease

Magnetic iron-oxide nanoparticles in the brain connected to alcohol-associated liver disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Magnetic iron-oxide nanoparticles in the brain connected to alcohol-associated liver disease Leon Kaub, Stefan Milz, Nirav Barapatre, Andreas Büttner, Bernhard Michalke, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4731478/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Magnetic iron-oxide nanoparticles in the form of magnetite (Fe3O4) are present in the human brain. They have been hypothesized to biomineralize in situ, as a result of dysfunctional iron homeostasis related to Alzheimer’s disease, or to enter the brain as airborne pollution particles. Regardless of their origin, magnetic iron-oxides pose a potential hazard to human health due to their high redox activity and surface charge. Here we report measurements on four post-mortem human brainstems, with one brainstem showing approximately 100 times higher magnetite concentrations than the other cases. This brainstem came from a subject with alcohol-associated liver disease (ALD) that manifested in liver cirrhosis and massive hepatic iron overload. Laser ablation – inductively coupled plasma – mass spectrometry showed the highest levels of trace metals (iron, copper and manganese) in the ALD brainstem. It is well established that a dysfunctional liver can result in the accumulation of trace metals in the brain. Our data indicate a similar pathway for magnetite particles, yet liver pathology has not been linked to magnetite occurrence in the brain so far. It may prove to be a crucial factor in understanding the high variation of magnetite concentrations found in human brains. Health sciences/Gastroenterology/Hepatology/Liver diseases/Alcoholic liver disease Biological sciences/Biochemistry/Metals/Iron Physical sciences/Nanoscience and technology/Nanotoxicology/Medical toxicology Biological sciences/Neuroscience Physical sciences/Materials science/Nanoscale materials/Magnetic properties and materials brain magnetite brain iron iron overload LA-ICP-MS alcohol use disorder Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Iron in the brain serves important roles for many biochemical processes, including oxygen transport, oxidative phosphorylation, myelination and the synthesis of neurotransmitters 1 . Most iron in the brain is bound to either hemoglobin, ferritin, hemosiderin or transferrin 2 . In these forms, iron does not carry a magnetic remanence 3,4 . Nevertheless, the presence of ferrimagnetic iron-oxide nanoparticles in the form of magnetite (Fe 3 O 4 ) in the brain was confirmed in multiple studies 5–8 . Their origin, potential physiological function and pathological risk are topics of ongoing research. The detection of well-shaped, euhedral crystals that resembled magnetite particles formed in magnetotactic bacteria led to the hypothesis that endogenously formed magnetite may provide a base for magnetoreception in humans 8,9 . In contrast, exogenous, pollution-based magnetite with high-temperature, combustion-derived crystal morphologies co-associated with non-physiological metals was found in the brain 5,7,10 . These magnetite particles were proposed to enter the brain through the olfactory nerve pathway 7 . Magnetite particles were also detected in cores of amyloid-β plaques, the main form of senile plaques in Alzheimer’s disease (AD), and have therefore been associated with dysfunctional iron homeostasis 11,12 . However, two recent studies showed no significant difference in magnetite concentrations between AD and non-AD brain tissue 5,13 . In the present study, we investigated the magnetic properties of four post-mortem human brainstems using superconducting quantum interference device (SQUID) magnetometry. We also examined iron-stained liver tissue from the corresponding subjects histologically since the liver plays a major role in iron metabolism 14 . Moreover, we employed laser ablation – inductively coupled plasma – mass spectrometry (LA-ICP-MS) to measure iron, copper and manganese in the brainstems, since a lack of hepatic metal removal in a dysfunctional liver can result in the accumulation of trace metals in the brain 15 . Results The brainstem samples from one case (141/22) had almost two orders of magnitude higher magnetic moments compared to three other cases (Fig. 1a and Supplementary Table 1). This was statistically significant for both natural remanent magnetizations (NRM) (p < 0.004) and saturated isothermal remanent magnetizations (SIRM) (p < 0.002) according to two-sided Mann-Whitney U tests. Most NRM, including that of case 141/22, and all SIRM exceeded the noise threshold of the magnetometer (Supplementary Fig. 1). The SIRM of the pons were significantly weaker than those of both the medulla oblongata (p = 0.023) and mesencephalon (p = 0.039) according to two-sided Wilcoxon signed-rank tests (Fig. 1b). No correlation was found between the magnetic moments and the age or sex of the subjects (Fig. 1a and Supplementary Table 2). Perl’s Prussian blue stained liver sections revealed iron overload only in the liver of case 141/22 (Fig. 1c). Iron deposition in the liver of case 141/22 followed a distinctive pattern (Supplementary Fig. 2) that led to the diagnosis of a secondary, non-hemochromatosis, iron overload disorder 16 and is characteristic of alcohol-associated liver disease (ALD) 17 : a heterogeneous distribution from one lobule to another; lack of iron within fibrous septa, biliary cells and vascular walls; and iron loading of parenchymal cells and Kupffer cells. Further supporting the diagnosis of ALD for case 141/22 was severe liver cirrhosis, which was most likely caused by chronic alcohol abuse 18 . The livers of the three other cases showed no signs of cirrhosis or iron deposition, as can be expected from normal hepatic iron conditions 16 (Fig. 1c). We further measured iron distributions in brain sections from all four cases with LA-ICP-MS (Fig. 2). The distributions covered parts of the Substantia Nigra pars compacta (SNpc) and the Red Nucleus (RN). In all four cases, the highest iron intensities were observed in the white matter region in between SNpc and RN. The ALD case 141/22 had the highest overall iron and copper levels (Fig. 3). Mean and maximum iron levels corresponded to SIRM values (Supplementary Fig. 3). The two iron isotopes as well as 63 Cu colocalized well in all four cases. 56 Fe had a consistently higher signal-to-noise ratio than 57 Fe, as expected given that 56 Fe and 57 Fe make up 91.8% and 2.1% of the iron isotopic composition in body tissue 19 . Parts of the oculomotor nerve identified as highly myelinated nerve fibers passing through the white matter between SNpc and RN were found in some of the investigated samples. For case 141/22, the oculomotor nerve colocalized with the main intensity peaks in all measured isotopes (Fig. 4). None of the other cases showed such colocalization (Fig. 2). Two round structures with uneven surfaces were identified as polymerization artefacts most likely caused by local excessive temperatures during polymerization (black arrows in Fig. 4). The upper artefact partly colocalized with iron, but not with copper or manganese. The lower artefact did not show a colocalization with any isotope. The artefacts therefore did not cause the isotope signals, and the maximum seen in the intensity distributions in all four isotopes can be attributed to oculomotor nerve fibers. 55 Mn had the highest noise levels of all four isotopes. For case 141/22, a peak in 55 Mn was observed in the oculomotor fibers (Fig. 4). Given that this 55 Mn signal colocalizes with 56 Fe, 57 Fe and 63 Cu, it is considered a robust signal. No comparable manganese signals were found in the three other cases. Discussion Magnetite nanoparticles have been detected within the core of amyloid- β plaques, leading to the hypothesis that they are associated with AD 11,12 . However, the four cases of the present study, including 141/22, did not show any signs of neuropathological conditions, thus excluding amyloid-β plaques as potential explanation for the high concentrations of magnetite in 141/22. Magnetite in the brain has also been hypothesized to originate from exogenous, pollution-based particles 5,7,10 . However, individuals from a city with extremely high air-pollution levels (Mexico City, Mexico) were reported with only five times higher magnetite concentrations in the brain compared to individuals from Manchester (UK) 5,10 . It is therefore unlikely that the observed differences in magnetite concentrations were caused by a difference in air-pollution exposure, particularly given that the samples were collected in Germany, where air-pollution levels are in general relatively low compared to Mexico City. Instead, we found case 141/22 to show massive hepatic iron overload and a severely damaged liver, which is characteristic of ALD (Fig. 1c). Liver failure can result in elevated systemic iron levels through a number of mechanisms, including decreased hepcidin expression, elevated transferrin and ferritin saturation and lack of removal of excess iron from the labile iron pool 14,20 . Furthermore, liver damage can cause increased iron, copper and manganese levels in the brain 21–24 . These trace metals influence the cellular redox balance and participate in the formation of reactive oxygen species 25 and can therefore subsequently lead to hepatic encephalopathy 15 . We found increased levels of these trace metals in the ALD brainstem, which aligns with the described mechanisms for liver damage 21–24 . For ALD in particular, increased iron concentrations in the brain is a known pathogenesis 23 . In addition, chronic alcohol abuse can lead to a disruption of the blood-brain barrier 26 . The LA-ICP-MS data further showed systematic distributions with maximum intensities in the white matter in between the SNpc and RN. Iron in the brain is generally more abundant in white matter than gray matter 27,28 . Even in nuclei that are typically identified as iron-enriched (such as the basal ganglia, the cerebellar nuclei and the SN), the cells with the highest iron levels are the myelin-forming oligodendrocytes 28,29 . With regard to the SN, iron in healthy individuals is mainly located in the Substantia Nigra reticulata (SNr), which is extensively myelinated by large amounts of oligodendrocytes, whereas the SNpc is not enriched in iron in healthy individuals 28 . This has recently been confirmed with LA-ICP-MS of rodent brain sections 30 . Given that the SNr is situated ventral to the SNpc, the LA-ICP-MS scans of this study did not cover the iron-enriched SNr. The investigated tissue samples also showed no signs of Parkinson’s disease, where substantially increased iron in neuromelanin-rich regions of the SNpc would be a typical finding 1 . Copper in the brain is also typically stored in glial cells and predominantly found in the locus coeruleus and the SN 31 . It is also linearly correlated with iron in physiological conditions 32 , which is confirmed by our data (Fig. 3). We further found the only valid manganese signal in the ALD case. Increased manganese concentrations in the SN are a typical finding in hepatic encephalopathy 21 . We detected manganese in the oculomotor nerve fibers that passed next to the SN (Fig. 4). These fibers also showed the highest iron and copper signals. Fibers from the oculomotor nerve showed excessive iron deposition in the case of progressive supranuclear palsy 33 . No signs of progressive supranuclear palsy were present for case 141/22, and whether ALD leads to a specific accumulation of iron in the oculomotor nerve is unknown. However, symptoms of chronic alcohol abuse include oculomotor signs as part of Wernicke’s syndrome 34 . Also, the oculomotor nerve is heavily myelinated, and myelin-formation in oligodendrocytes requires high amounts of iron 29 . The ALD case of the present study showed typical hepatic iron overload and elevated levels of iron, copper and manganese in the brain, which was not unexpected given that trace metals and in particular iron can accumulate in the liver and the brain of ALD patients. We also found exceedingly high concentrations of magnetite nanoparticles in the brain tissue of the ALD case. We therefore suggest that a damaged liver with concurrent hepatic iron overload, such as in ALD, can cause magnetite nanoparticles to accumulate in the brain. Considering these findings, a distribution towards the brainstem and cerebellum (as found by ref. 6 ) may be attributed to the blood supply to the brain since the vertebrobasilar system, which supplies the posterior brain with oxygen-rich blood, runs directly along the brainstem. Our hypothesis is also supported by the repeated findings that meninges show high concentrations of magnetite 8,35 . The origin of the magnetite particles in the body remains unknown, but both exogenous and endogenous particles could be transported to the brain in blood. So far, exogenous particles were suggested to reach the brain through the olfactory bulb 5,7 or the neuroenteric system and the vagal nerve 10 . However, the olfactory nerve enters the brain in the frontal lobe, where relatively low concentrations of magnetite were found 6 . On the other hand, pollution-based magnetite particles may get into the blood stream by a take-up through enterocytes or penetration through the respiratory epithelial barrier. Similarly, endogenous magnetite particles may not be formed in the brain but in other organs, such as the liver, which has been shown to have elevated magnetite concentrations 35 . Collectively, the findings of this study indicate that magnetite particles can accumulate in the human brain as a consequence of liver damage. This mechanism has not been considered for magnetite occurrence in the brain so far. Several authors reported large variations in magnetite concentrations in brain tissue, and speculations about the underlying reasons ranged from hypothetically different levels of air-pollution exposure to schizophrenia 5,6 . However, potential concomitant liver pathology has not yet been considered in any study on magnetite in the brain, and thus, these hypotheses 5,6 should be interpreted cautiously. The results presented here clearly indicate that future research on magnetite in the brain should consider liver pathology. Materials and Methods Brain tissue samples Post-mortem brainstems from four subjects were collected at the Institute of Forensic Medicine at the University of Rostock (Germany). A forensic autopsy was performed as requested by the public prosecutor and approved by a judicial decision. For this type of autopsy, formal consent is not required. Furthermore, the study was approved by the Local Ethical Committee at the Medical University of Rostock (ethical approval number: A 2021-0282). All work was conducted in accordance with official regulations and guidelines by the state of Mecklenburg-Vorpommern, Germany. Age, sex, the cause of death and the post-mortem interval were known for each individual, which otherwise remained anonymous (Supplementary Table 1). The individuals died for reasons unrelated to the brain, and the four brains showed no signs of neurological damage. Extraction followed standard procedures, with much attention paid to minimize the risk of contaminating the tissue with magnetic particles. Solely the opening of the skull was done with a metallic saw, but care was taken not to damage the dura mater. During the skull opening, the brainstem was protected from potential magnetic contaminants by the rest of the overlying brain. For the following extraction and dissections, only ceramic tools that had been washed with 10% HCl and filtered distilled water were used. Immediately after extraction, the brainstems were stored in sterile plastic bags at -20°C. The four brainstems were immersion fixed in buffered, filtered 10% formaldehyde for five days. During fixation, daily measurements of the pH-value were taken to monitor formaldehyde acidity. The formaldehyde was replaced twice during fixation to ensure a stable pH-value of 7.0. This procedure should not have altered the magnetic properties of the tissue 13 . Even if fixation lowered magnetite concentrations, magnetic moments would be systematically lowered in all brainstems since they were treated identically. After fixation, the dura and arachnoid mater as well as large blood vessels were carefully removed. Each brainstem was divided by two horizontal cuts (separating the medulla oblongata, pons and mesencephalon) and one sagittal cut (separating the left and right portions of each structure), resulting in six tissue samples per brainstem. Tissue samples were weighed and stored in sterile plastic cups, which were washed with 10% HCl and filtered distilled water. Autopsy, fixation, cutting and storage protocols of all four brainstems were identical and performed on the same day except for the autopsy, which took place in the preceding weeks. Liver tissue samples from biopsies were embedded in paraffin following standard protocols. For each case, one 5 µm-thin paraffin section was stained with hematoxylin-eosin (HE) and a second one with Perl’s Prussian blue. Measurements of Magnetic Moments Magnetic moments of the tissue samples were measured following existing protocols 6 . In short, the full vector magnetic moment of each sample was measured with a three-axis superconducting magnetometer (2G Enterprises Inc., Mountain View, CA, USA) at room temperature. Tissue samples were first measured in their unmagnetized, natural state (natural remanent magnetization, NRM). They were then exposed to a 0.63 T magnetic field using an electromagnet, followed by measurement of their acquired saturated isothermal remanent magnetization (SIRM). Each measurement consisted of four recordings, with the tissue sample being rotated 90° four times in the x-y plane. The four recordings were averaged, and baseline measurements (without tissue sample) subtracted. The magnetic moment measurements were repeated 22 times for different samples in order to define the instrument noise level. Magnetic moments were recorded using CryoMag software 36 . The magnetometer was situated in a magnetically shielded room (approximately 200 nT ambient field) in a forest 80 km northeast of Munich (Germany). In addition to fewer anthropogenic magnetic particles in the air of a forest compared to urban environments 37 , the magnetically shielded room was converted to a clean room before measurements to minimize the risk of magnetic contamination. Much effort was taken to perform the measurements in a magnetically clean environment: (i) inflowing air was filtered (high efficiency particulate air [HEPA] filter and electromagnetic filter); (ii) only one person handled samples and the sample holder while a second person operated the measuring system; (iii) both persons wore personal clean room equipment; and (iv) the exposure of samples to air was kept to a minimum (about two minutes for each measurement). Laser Ablation – Inductively Coupled Plasma – Mass Spectrometry (LA-ICP-MS) Distributions of iron, copper and manganese in the brain tissue were measured using LA-ICP-MS. Data were recorded on one mesencephalon sample from each case (157/22, left hemisphere; other cases, right hemisphere). LA-ICP-MS measured the two-dimensional distribution of selected isotopes by ablating particles from the sample surface with a laser, which were directed to ICP-MS by an Argon gas flow and analyzed. To achieve plane sample surfaces required by this method, mesencephalon samples from each of the four brainstems were embedded in methylmethacrylate (MMA) according to existing protocols 38,39 . To this end, the samples were first dehydrated in ethanol baths of increasing concentration (70, 80, 90, 100%), then degreased in xylene, followed by incubation in 100% methanol. Finally, the samples were embedded in MMA (product number: 800590, Sigma Aldrich, St. Louis, MO, USA). To minimize contamination with magnetic particles, chemicals were filtered with PTFE filters (mesh size, 0.05 µm) except for more viscous chemicals that were filtered with a 0.2 µm mesh. The chemicals were filtered into glasses that were pre-washed with 10% HCl and rinsed with filtered distilled water. Slices of approximately 600 µm in thickness were cut from the MMA blocks using a circular saw microtome (SP 1600, Leica, Wetzlar, Germany). The slices were ground and polished with a 400 CS micro-grinder (EXAKT Advanced Technologies, Norderstedt, Germany), which decreased the slice thicknesses to approximately 400 µm; surfaces were thoroughly cleaned with isopropanol after polishing. The LA-ICP-MS instrument consisted of a laser ablation system (NWR-213, New Wave Research Inc., Fremont, CA, USA) coupled to a NexION 300 ICP-MS (PerkinElmer, Waltham, MA, USA), with distributions of two iron isotopes ( 56 Fe, 57 Fe), copper ( 63 Cu) and manganese ( 55 Mn) being continuously recorded (Supplementary Table 3). For each sample, an area of 5 x 1.7 mm was selected covering parts of the Substantia nigra pars compacta (SNpc), which was easily visible due to large amounts of cells containing neuromelanin. Besides this being an area of interest for metals in the brain, the neuromelanin was used for orientation since the samples remained unstained for LA-ICP-MS to avoid possible contamination from staining. The scanned area was selected to extend from the SNpc to the red nucleus (RN) when possible. For case 159/22, the area was increased to 6 x 1.7 mm to scan a larger portion of the RN. The scans were oriented in a similar way for each sample, with the SNpc on the left side of the scanned area (i.e., during each line, the laser ablated first from the SNpc, then from white matter and lastly from the RN). Scans were done line-wise with a total of 67 lines per scan; intensities were uncalibrated. For each scan, data from the first ten seconds of each line were discarded due to the delay between laser ablation and ICP-MS measurement; data were analyzed using Laser Ablation App 1.0 (unpublished). Light Microscopical Analysis Following LA-ICP-MS, the brainstem sections were stained with Toluidine blue and imaged using a digital single-lens reflex camera (EOS 5D, Canon Inc., Ōta, Tokyo, Japan) equipped with a macro objective (LM macroscope 42x XL, Micro Tech Lab, Graz, Austria). Stained liver tissue samples were imaged with a light microscope (Axio imager.M2, ZEISS, Oberkochen, Germany) equipped with a 40x objective (Plan-Apochromat 40x, ZEISS, Germany) and operated with Stereo Investigator software (MBF Bioscience, Williston, VT, USA). Images were analyzed using an image browser (Biolucida Viewer, MBF Bioscience). Figures were constructed using a raster graphics editor (Affinity Photo 2, Serif Europe Ltd., Nottingham, UK). Only contrast and brightness adjustments were made with this editor, without altering the appearance of the original materials. Statistical analysis Data were analyzed with customized scripts in Python programming language (version 3.9, Python Software Foundation, Wilmington, DE, USA). Statistical tests included Mann-Whitney-U tests for independent samples and Wilcoxon signed-rank tests for dependent samples, both using confidence levels of 95%. References 1. Ward, R. J., Zucca, F. A., Duyn, J. H., Crichton, R. R. & Zecca, L. 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Quantitative morphology of the subchondral plate of the tibial plateau. J. Anat. 185, 103–110 (1994). 39. He, X. et al. Analysis of titanium and other metals in human jawbones with dental implants - A case series study. Dent. Mater. 32, 1042–1051; 10.1016/j.dental.2016.05.012 (2016). 40. Kaub, L. et al. Magnetic and LA-ICP-MS data for magnetic iron-oxide nanoparticles in the brain connected to alcohol-associated liver disease. figshare https://doi.org/10.6084/m9.figshare.25908568 (2024). Declarations Acknowledgements We thank Hans-Georg Frank for helpful advice and Claudia Harbauer, Andrea Haderer, Beate Aschauer and Manuela Weiß for skillful technical assistance. Laser Ablation App 1.0 was kindly provided by Daniel Hemmler. This work was supported by the German Research Foundation (DFG project GI712-25/1). Author contributions L.K., S.M., C.S. and S.A.G. designed research; L.K., S.M., A.B., B.M. and S.A.G. performed research; L.K. and S.A.G. analyzed data; L.K., S.M., N.B., C.S. and S.A.G. interpreted results; and L.K., C.S. and S.A.G. wrote the paper and all authors read and contributed comments to the work. Data Availability Statement All source data are available on the figshare repository at https://doi.org/10.6084/m9.figshare.25908568 (ref. 40 ). Competing Interests Statement The authors declare no competing interests. Additional Declarations No competing interests reported. Supplementary Files SciRepSupplementaryMaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 08 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 25 Nov, 2024 Reviews received at journal 20 Nov, 2024 Reviewers agreed at journal 08 Nov, 2024 Reviews received at journal 29 Oct, 2024 Reviewers agreed at journal 17 Oct, 2024 Reviewers agreed at journal 06 Aug, 2024 Reviewers invited by journal 30 Jul, 2024 Editor assigned by journal 30 Jul, 2024 Editor invited by journal 27 Jul, 2024 Submission checks completed at journal 24 Jul, 2024 First submitted to journal 12 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4731478","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":336850656,"identity":"fa87fd23-4a28-4bdd-9395-4ee03bad29a4","order_by":0,"name":"Leon Kaub","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACPjB5gIHBgL25AchiJqyFDa6F5yDJWiQSidXC3nzsAcMZu8Ttkg9bN/MwWMsR1sJzLN2A4UZy4s7ZiW23eRjSjQlrkcgxk2D4wJy44TZYy2Gw8/BrkX8D0lKfuOHmQbCWesJaJHiAWm4cTtxwgxGsJYEIv6SlSSScOW68syex7eYcg3RDgrbwsx8+JvHhWLXsdiDjxpsKa3mCtoABwjEGxGkYBaNgFIyCUUAAAAATYTvPJo6ArwAAAABJRU5ErkJggg==","orcid":"","institution":"Ludwig-Maximilians-University of Munich","correspondingAuthor":true,"prefix":"","firstName":"Leon","middleName":"","lastName":"Kaub","suffix":""},{"id":336850657,"identity":"e1b0b6dd-cf45-4d9b-b9fc-61f1d329f615","order_by":1,"name":"Stefan Milz","email":"","orcid":"","institution":"Ludwig-Maximilians-University of Munich","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Milz","suffix":""},{"id":336850658,"identity":"7224b9af-d775-4326-be15-350a34d05d75","order_by":2,"name":"Nirav Barapatre","email":"","orcid":"","institution":"Ludwig-Maximilians-University of Munich","correspondingAuthor":false,"prefix":"","firstName":"Nirav","middleName":"","lastName":"Barapatre","suffix":""},{"id":336850659,"identity":"f633085f-fc61-453c-9fd7-22754e53fa42","order_by":3,"name":"Andreas Büttner","email":"","orcid":"","institution":"University of Rostock","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Büttner","suffix":""},{"id":336850660,"identity":"7cee899d-38cf-4bf4-90ae-33847e3856fd","order_by":4,"name":"Bernhard Michalke","email":"","orcid":"","institution":"Helmholtz Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Bernhard","middleName":"","lastName":"Michalke","suffix":""},{"id":336850661,"identity":"0d6bc419-4218-4c8c-b69c-c87d719a6e25","order_by":5,"name":"Christoph Schmitz","email":"","orcid":"","institution":"Ludwig-Maximilians-University of Munich","correspondingAuthor":false,"prefix":"","firstName":"Christoph","middleName":"","lastName":"Schmitz","suffix":""},{"id":336850662,"identity":"a88d1b2d-d407-4f9e-aff0-de3f548b4138","order_by":6,"name":"Stuart A. Gilder","email":"","orcid":"","institution":"Ludwig-Maximilians-University of Munich","correspondingAuthor":false,"prefix":"","firstName":"Stuart","middleName":"A.","lastName":"Gilder","suffix":""}],"badges":[],"createdAt":"2024-07-12 16:02:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4731478/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4731478/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-09756-8","type":"published","date":"2025-07-08T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62406476,"identity":"28ccf443-c0d5-430b-afab-b30c1867b42f","added_by":"auto","created_at":"2024-08-13 21:11:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":377098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMagnetometry results and pathohistological examination of liver tissue. a\u003c/strong\u003e Mass normalized natural remanent magnetization (NRM) and saturated isothermal remanent magnetization (SIRM) of the four investigated brainstems, ordered by age of the subjects. Data are represented as mean magnetic moments with 95% confidence intervals as well as individual data points. \u003cstrong\u003eb\u003c/strong\u003e Regional (vertical) distribution of mass normalized SIRM throughout the brainstems. Data are represented as median SIRM of each structure (black lines) and individual data points, *p \u0026lt; 0.05 according to two-sided Wilcoxon signed-rank tests. \u003cstrong\u003ec\u003c/strong\u003e Perl’s Prussian blue stained liver tissue from each case (also see Supplementary Fig. 2). Scale bar: 200 µm.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4731478/v1/46028325da22eb809b9178cc.jpeg"},{"id":62406243,"identity":"723eefe2-e7d4-42cd-9544-5041645462f3","added_by":"auto","created_at":"2024-08-13 21:03:05","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":744761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron distribution measured by LA-ICP-MS in mesencephalon samples from each case.\u003c/strong\u003e \u003cstrong\u003ea-c\u003c/strong\u003e 141/22; \u003cstrong\u003ed-f\u003c/strong\u003e 144/22; \u003cstrong\u003eg-h\u003c/strong\u003e 157/22; \u003cstrong\u003ej-l\u003c/strong\u003e 159/22. \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e Images of the full sections and drawings illustrate the orientation of the samples as well as the fields of view (dashed rectangles) shown in the other panels. The overview of case 157/22 (\u003cstrong\u003eg\u003c/strong\u003e) was horizontally mirrored to show correct orientation. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e Photomicrographs showing the structures covered by LA-ICP-MS. Highly visible accumulations of neuromelanin-containing cells as part of the Substantia Nigra pars compacta (SNpc) were observed in each sample. The red nucleus (RN), identified as the oval region dorsomedial from the SNpc with higher cell densities and fewer nerve fibers than surrounding areas, is illustrated by a dashed line in all panels. Fibers of the oculomotor nerve are marked in continuous ovals. The sample for case 144/22 was cut more caudal leading to no oculomotor fibers and causing the RN to appear smaller and with a larger distance to the SNpc. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e \u003csup\u003e56\u003c/sup\u003eFe intensity distributions, with color scales adapted for each scan (arbitrary units). LA-ICP-MS scans (rectangles) covered an area of 1.7 x 5 mm (\u003cstrong\u003ea-i\u003c/strong\u003e) or 1.7 x 6 mm (\u003cstrong\u003ej-l\u003c/strong\u003e). Abbreviations: SCN, superior colliculus nucleus; CA, cerebral aqueduct; ON, oculomotor nucleus; RF, reticular formation; CP, cerebral peduncle; III, third cranial (oculomotor) nerve.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4731478/v1/993e93ab61e504091ff893a5.jpeg"},{"id":62406245,"identity":"f156c7fe-924c-4b48-9a3b-686673bdc947","added_by":"auto","created_at":"2024-08-13 21:03:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1191785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwo-dimensional distributions of iron (\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e56\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eFe and \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e57\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eFe), copper (\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e63\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCo) and manganese (\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e55\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eMn) measured by LA-ICP-MS for each case.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e 141/22;\u003cstrong\u003e b\u003c/strong\u003e 144/22; \u003cstrong\u003ec\u003c/strong\u003e 157/22; and \u003cstrong\u003ed\u003c/strong\u003e 159/22. Intensities were normalized by the mean intensity (µ) of each distribution (given in lower left corner). Black rectangles illustrate the scanned regions for case 141/22 shown at higher magnification in Fig. 4.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4731478/v1/c4ec2e3a8bf8125904584d99.jpeg"},{"id":62406241,"identity":"e0e6a5b2-81d4-49b1-a285-4359d116499b","added_by":"auto","created_at":"2024-08-13 21:03:05","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":604508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh-magnification details of the LA-ICP-MS scans of case 141/22\u003c/strong\u003e (indicated by black rectangles in Fig. 3a). \u003cstrong\u003ea\u003c/strong\u003e Microphotograph of the Toluidin-blue stained section (Scale bar: 200 µm). \u003cstrong\u003eb-e\u003c/strong\u003e LA-ICP-MS data of (\u003cstrong\u003eb\u003c/strong\u003e) \u003csup\u003e56\u003c/sup\u003eFe, (\u003cstrong\u003ec\u003c/strong\u003e) \u003csup\u003e57\u003c/sup\u003eFe, (\u003cstrong\u003ed\u003c/strong\u003e) \u003csup\u003e63\u003c/sup\u003eCu and (\u003cstrong\u003ee\u003c/strong\u003e) \u003csup\u003e55\u003c/sup\u003eMn of the corresponding region. Intensities were normalized by the mean intensity of each scan (see also Fig. 3). Myelinated fibers from the oculomotor nerve passed through this region (black ovals). Two round polymerization artefacts (black arrows) did not colocalize with the isotope distributions.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4731478/v1/e3f299efe9ff1005c18dd015.jpeg"},{"id":86699288,"identity":"06f80d40-cd8c-444f-97eb-b65fd254825c","added_by":"auto","created_at":"2025-07-14 16:07:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3689870,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4731478/v1/164cfce1-0496-463e-bb36-dc9ce39730e5.pdf"},{"id":62406721,"identity":"4280507c-b8e0-4956-b0b6-cc6f937ff20b","added_by":"auto","created_at":"2024-08-13 21:19:05","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":657213,"visible":true,"origin":"","legend":"","description":"","filename":"SciRepSupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4731478/v1/cc85a59a6f2beca703b8fa6b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Magnetic iron-oxide nanoparticles in the brain connected to alcohol-associated liver disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIron in the brain serves important roles for many biochemical processes, including oxygen transport, oxidative phosphorylation, myelination and the synthesis of neurotransmitters\u003csup\u003e1\u003c/sup\u003e. Most iron in the brain is bound to either hemoglobin, ferritin, hemosiderin or transferrin\u003csup\u003e2\u003c/sup\u003e. In these forms, iron does not carry a magnetic remanence\u003csup\u003e3,4\u003c/sup\u003e. Nevertheless, the presence of ferrimagnetic iron-oxide nanoparticles in the form of magnetite (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) in the brain was confirmed in multiple studies\u003csup\u003e5\u0026ndash;8\u003c/sup\u003e. Their origin, potential physiological function and pathological risk are topics of ongoing research. The detection of well-shaped, euhedral crystals that resembled magnetite particles formed in magnetotactic bacteria led to the hypothesis that endogenously formed magnetite may provide a base for magnetoreception in humans\u003csup\u003e8,9\u003c/sup\u003e. In contrast, exogenous, pollution-based magnetite with high-temperature, combustion-derived crystal morphologies co-associated with non-physiological metals was found in the brain\u003csup\u003e5,7,10\u003c/sup\u003e. These magnetite particles were proposed to enter the brain through the olfactory nerve pathway\u003csup\u003e7\u003c/sup\u003e. Magnetite particles were also detected in cores of amyloid-\u0026beta; plaques, the main form of senile plaques in Alzheimer\u0026rsquo;s disease (AD), and have therefore been associated with dysfunctional iron homeostasis\u003csup\u003e11,12\u003c/sup\u003e. However, two recent studies showed no significant difference in magnetite concentrations between AD and non-AD brain tissue\u003csup\u003e5,13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the present study, we investigated the magnetic properties of four post-mortem human brainstems using superconducting quantum interference device (SQUID) magnetometry. We also examined iron-stained liver tissue from the corresponding subjects histologically since the liver plays a major role in iron metabolism\u003csup\u003e14\u003c/sup\u003e. Moreover, we employed laser ablation \u0026ndash; inductively coupled plasma \u0026ndash; mass spectrometry (LA-ICP-MS) to measure iron, copper and manganese in the brainstems, since a lack of hepatic metal removal in a dysfunctional liver can result in the accumulation of trace metals in the brain\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe brainstem samples from one case (141/22) had almost two orders of magnitude higher magnetic moments compared to three other cases (Fig. 1a and Supplementary Table 1). This was statistically significant for both natural remanent magnetizations (NRM) (p \u0026lt; 0.004) and saturated isothermal remanent magnetizations (SIRM) (p \u0026lt; 0.002) according to two-sided Mann-Whitney U tests. Most NRM, including that of case 141/22, and all SIRM exceeded the noise threshold of the magnetometer (Supplementary Fig. 1). The SIRM of the pons were significantly weaker than those of both the medulla oblongata (p = 0.023) and mesencephalon (p = 0.039) according to two-sided Wilcoxon signed-rank tests (Fig. 1b). No correlation was found between the magnetic moments and the age or sex of the subjects (Fig. 1a and Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003ePerl\u0026rsquo;s Prussian blue stained liver sections revealed iron overload only in the liver of case 141/22 (Fig. 1c). Iron deposition in the liver of case 141/22 followed a distinctive pattern (Supplementary Fig. 2) that led to the diagnosis of a secondary, non-hemochromatosis, iron overload disorder\u003csup\u003e16\u003c/sup\u003e and is characteristic of alcohol-associated liver disease (ALD)\u003csup\u003e17\u003c/sup\u003e: a heterogeneous distribution from one lobule to another; lack of iron within fibrous septa, biliary cells and vascular walls; and iron loading of parenchymal cells and Kupffer cells. Further supporting the diagnosis of ALD for case 141/22 was severe liver cirrhosis, which was most likely caused by chronic alcohol abuse\u003csup\u003e18\u003c/sup\u003e. The livers of the three other cases showed no signs of cirrhosis or iron deposition, as can be expected from normal hepatic iron conditions\u003csup\u003e16\u003c/sup\u003e (Fig. 1c).\u003c/p\u003e\n\u003cp\u003eWe further measured iron distributions in brain sections from all four cases with LA-ICP-MS (Fig. 2). The distributions covered parts of the Substantia Nigra pars compacta (SNpc) and the Red Nucleus (RN). In all four cases, the highest iron intensities were observed in the white matter region in between SNpc and RN.\u003c/p\u003e\n\u003cp\u003eThe ALD case 141/22 had the highest overall iron and copper levels (Fig. 3). Mean and maximum iron levels corresponded to SIRM values (Supplementary Fig. 3). The two iron isotopes as well as \u003csup\u003e63\u003c/sup\u003eCu colocalized well in all four cases. \u003csup\u003e56\u003c/sup\u003eFe had a consistently higher signal-to-noise ratio than \u003csup\u003e57\u003c/sup\u003eFe, as expected given that \u003csup\u003e56\u003c/sup\u003eFe and \u003csup\u003e57\u003c/sup\u003eFe make up 91.8% and 2.1% of the iron isotopic composition in body tissue\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eParts of the oculomotor nerve identified as highly myelinated nerve fibers passing through the white matter between SNpc and RN were found in some of the investigated samples. For case 141/22, the oculomotor nerve colocalized with the main intensity peaks in all measured isotopes (Fig. 4). None of the other cases showed such colocalization (Fig. 2). Two round structures with uneven surfaces were identified as polymerization artefacts\u0026nbsp;most likely caused by local excessive temperatures during polymerization (black arrows in Fig. 4). The upper artefact partly colocalized with iron, but not with copper or manganese. The lower artefact did not show a colocalization with any isotope. The artefacts therefore did not cause the isotope signals, and the maximum seen in the intensity distributions in all four isotopes can be attributed to oculomotor nerve fibers. \u003csup\u003e55\u003c/sup\u003eMn\u003csup\u003e\u0026nbsp;\u003c/sup\u003ehad the highest noise levels of all four isotopes. For case 141/22, a peak in \u003csup\u003e55\u003c/sup\u003eMn was observed in the oculomotor fibers (Fig. 4). Given that this \u003csup\u003e55\u003c/sup\u003eMn signal colocalizes with \u003csup\u003e56\u003c/sup\u003eFe, \u003csup\u003e57\u003c/sup\u003eFe and \u003csup\u003e63\u003c/sup\u003eCu, it is considered a robust signal. No comparable manganese signals were found in the three other cases.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMagnetite nanoparticles have been detected within the core of amyloid-\u0026nbsp;\u0026beta; plaques, leading to the hypothesis that they are associated with AD\u003csup\u003e11,12\u003c/sup\u003e. However, the four cases of the present study, including 141/22, did not show any signs of neuropathological conditions, thus excluding amyloid-\u0026beta; plaques as potential explanation for the high concentrations of magnetite in 141/22. Magnetite in the brain has also been hypothesized to originate from exogenous, pollution-based particles\u003csup\u003e5,7,10\u003c/sup\u003e. However, individuals from a city with extremely high air-pollution levels (Mexico City, Mexico) were reported with only five times higher magnetite concentrations in the brain compared to individuals from Manchester (UK)\u003csup\u003e5,10\u003c/sup\u003e. It is therefore unlikely that the observed differences in magnetite concentrations were caused by a difference in air-pollution exposure, particularly given that the samples were collected in Germany, where air-pollution levels are in general relatively low compared to Mexico City.\u003c/p\u003e\n\u003cp\u003eInstead, we found case 141/22 to show massive hepatic iron overload and a severely damaged liver, which is characteristic of ALD (Fig. 1c). Liver failure can result in elevated systemic iron levels through a number of mechanisms, including decreased hepcidin expression, elevated transferrin and ferritin saturation and lack of removal of excess iron from the labile iron pool\u003csup\u003e14,20\u003c/sup\u003e. Furthermore, liver damage can cause increased iron, copper and manganese levels in the brain\u003csup\u003e21\u0026ndash;24\u003c/sup\u003e. These trace metals influence the cellular redox balance and participate in the formation of reactive oxygen species\u003csup\u003e25\u003c/sup\u003e and can therefore subsequently lead to hepatic encephalopathy\u003csup\u003e15\u003c/sup\u003e. We found increased levels of these trace metals in the ALD brainstem, which aligns with the described mechanisms for liver damage\u003csup\u003e21\u0026ndash;24\u003c/sup\u003e. For ALD in particular, increased iron concentrations in the brain is a known pathogenesis\u003csup\u003e23\u003c/sup\u003e. In addition, chronic alcohol abuse can lead to a disruption of the blood-brain barrier\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe LA-ICP-MS data further showed systematic distributions with maximum intensities in the white matter in between the SNpc and RN. Iron in the brain is generally more abundant in white matter than gray matter\u003csup\u003e27,28\u003c/sup\u003e. Even in nuclei that are typically identified as iron-enriched (such as the basal ganglia, the cerebellar nuclei and the SN), the cells with the highest iron levels are the myelin-forming oligodendrocytes\u003csup\u003e28,29\u003c/sup\u003e. With regard to the SN, iron in healthy individuals is mainly located in the Substantia Nigra reticulata (SNr), which is extensively myelinated by large amounts of oligodendrocytes, whereas the SNpc is not enriched in iron in healthy individuals\u003csup\u003e28\u003c/sup\u003e. This has recently been confirmed with LA-ICP-MS of rodent brain sections\u003csup\u003e30\u003c/sup\u003e. Given that the SNr is situated ventral to the SNpc, the LA-ICP-MS scans of this study did not cover the iron-enriched SNr. The investigated tissue samples also showed no signs of Parkinson\u0026rsquo;s disease, where substantially increased iron in neuromelanin-rich regions of the SNpc would be a typical finding\u003csup\u003e1\u003c/sup\u003e. Copper in the brain is also typically stored in glial cells and predominantly found in the locus coeruleus and the SN\u003csup\u003e31\u003c/sup\u003e. It is also linearly correlated with iron in physiological conditions\u003csup\u003e32\u003c/sup\u003e, which is confirmed by our data (Fig. 3). We further found the only valid manganese signal in the ALD case. Increased manganese concentrations in the SN are a typical finding in hepatic encephalopathy\u003csup\u003e21\u003c/sup\u003e. We detected manganese in the oculomotor nerve fibers that passed next to the SN (Fig. 4). These fibers also showed the highest iron and copper signals. Fibers from the oculomotor nerve showed excessive iron deposition in the case of progressive supranuclear palsy\u003csup\u003e33\u003c/sup\u003e. No signs of progressive supranuclear palsy were present for case 141/22, and whether ALD leads to a specific accumulation of iron in the oculomotor nerve is unknown. However, symptoms of chronic alcohol abuse include oculomotor signs as part of Wernicke\u0026rsquo;s syndrome\u003csup\u003e34\u003c/sup\u003e. Also, the oculomotor nerve is heavily myelinated, and myelin-formation in oligodendrocytes requires high amounts of iron\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe ALD case of the present study showed typical hepatic iron overload and elevated levels of iron, copper and manganese in the brain, which was not unexpected given that trace metals and in particular iron can accumulate in the liver and the brain of ALD patients. We also found exceedingly high concentrations of magnetite nanoparticles in the brain tissue of the ALD case. We therefore suggest that a damaged liver with concurrent hepatic iron overload, such as in ALD, can cause magnetite nanoparticles to accumulate in the brain. Considering these findings, a distribution towards the brainstem and cerebellum (as found by ref.\u003csup\u003e6\u003c/sup\u003e) may be attributed to the blood supply to the brain since the vertebrobasilar system, which supplies the posterior brain with oxygen-rich blood, runs directly along the brainstem. Our hypothesis is also supported by the repeated findings that meninges show high concentrations of magnetite\u003csup\u003e8,35\u003c/sup\u003e. The origin of the magnetite particles in the body remains unknown, but both exogenous and endogenous particles could be transported to the brain in blood. So far, exogenous particles were suggested to reach the brain through the olfactory bulb\u003csup\u003e5,7\u003c/sup\u003e or the neuroenteric system and the vagal nerve\u003csup\u003e10\u003c/sup\u003e. However, the olfactory nerve enters the brain in the frontal lobe, where relatively low concentrations of magnetite were found\u003csup\u003e6\u003c/sup\u003e. On the other hand, pollution-based magnetite particles may get into the blood stream by a take-up through enterocytes or penetration through the respiratory epithelial barrier. Similarly, endogenous magnetite particles may not be formed in the brain but in other organs, such as the liver, which has been shown to have elevated magnetite concentrations\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, the findings of this study indicate that magnetite particles can accumulate in the human brain as a consequence of liver damage. This mechanism has not been considered for magnetite occurrence in the brain so far. Several authors reported large variations in magnetite concentrations in brain tissue, and speculations about the underlying reasons ranged from hypothetically different levels of air-pollution exposure to schizophrenia\u003csup\u003e5,6\u003c/sup\u003e. However, potential concomitant liver pathology has not yet been considered in any study on magnetite in the brain, and thus, these hypotheses\u003csup\u003e5,6\u003c/sup\u003e should be interpreted cautiously. The results presented here clearly indicate that future research on magnetite in the brain should consider liver pathology.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eBrain tissue samples\u003c/h2\u003e\n\u003cp\u003ePost-mortem brainstems from four subjects were collected at the Institute of Forensic Medicine at the University of Rostock (Germany). A forensic autopsy was performed as requested by the public prosecutor and approved by a judicial decision. For this type of autopsy, formal consent is not required. Furthermore, the study was approved by the Local Ethical Committee at the Medical University of Rostock (ethical approval number: A 2021-0282). All work was conducted in accordance with official regulations and guidelines by the state of Mecklenburg-Vorpommern, Germany. Age, sex, the cause of death and the post-mortem interval were known for each individual, which otherwise remained anonymous (Supplementary Table 1). The individuals died for reasons unrelated to the brain, and the four brains showed no signs of neurological damage. Extraction followed standard procedures, with much attention paid to minimize the risk of contaminating the tissue with magnetic particles. Solely the opening of the skull was done with a metallic saw, but care was taken not to damage the dura mater. During the skull opening, the brainstem was protected from potential magnetic contaminants by the rest of the overlying brain. For the following extraction and dissections, only ceramic tools that had been washed with 10% HCl and filtered distilled water were used. Immediately after extraction, the brainstems were stored in sterile plastic bags at -20\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe four brainstems were immersion fixed in buffered, filtered 10% formaldehyde for five days. During fixation, daily measurements of the pH-value were taken to monitor formaldehyde acidity. The formaldehyde was replaced twice during fixation to ensure a stable pH-value of 7.0. This procedure should not have altered the magnetic properties of the tissue\u003csup\u003e13\u003c/sup\u003e. Even if fixation lowered magnetite concentrations, magnetic moments would be systematically lowered in all brainstems since they were treated identically. After fixation, the dura and arachnoid mater as well as large blood vessels were carefully removed. Each brainstem was divided by two horizontal cuts (separating the medulla oblongata, pons and mesencephalon) and one sagittal cut (separating the left and right portions of each structure), resulting in six tissue samples per brainstem. Tissue samples were weighed and stored in sterile plastic cups, which were washed with 10% HCl and filtered distilled water. Autopsy, fixation, cutting and storage protocols of all four brainstems were identical and performed on the same day except for the autopsy, which took place in the preceding weeks.\u003c/p\u003e\n\u003cp\u003eLiver tissue samples from biopsies were embedded in paraffin following standard protocols. For each case, one 5 \u0026micro;m-thin paraffin section was stained with hematoxylin-eosin (HE) and a second one with Perl\u0026rsquo;s Prussian blue.\u003c/p\u003e\n\u003ch2\u003eMeasurements of Magnetic Moments\u003c/h2\u003e\n\u003cp\u003eMagnetic moments of the tissue samples were measured following existing protocols\u003csup\u003e6\u003c/sup\u003e. In short, the full vector magnetic moment of each sample was measured with a three-axis superconducting magnetometer (2G Enterprises Inc., Mountain View, CA, USA) at room temperature. Tissue samples were first measured in their unmagnetized, natural state (natural remanent magnetization, NRM). They were then exposed to a 0.63 T magnetic field using an electromagnet, followed by measurement of their acquired saturated isothermal remanent magnetization (SIRM). Each measurement consisted of four recordings, with the tissue sample being rotated 90\u0026deg; four times in the x-y plane. The four recordings were averaged, and baseline measurements (without tissue sample) subtracted. The magnetic moment measurements were repeated 22 times for different samples in order to define the instrument noise level. Magnetic moments were recorded using CryoMag software\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe magnetometer was situated in a magnetically shielded room (approximately 200 nT ambient field) in a forest 80 km northeast of Munich (Germany). In addition to fewer anthropogenic magnetic particles in the air of a forest compared to urban environments\u003csup\u003e37\u003c/sup\u003e, the magnetically shielded room was converted to a clean room before measurements to minimize the risk of magnetic contamination. Much effort was taken to perform the measurements in a magnetically clean environment: (i)\u0026nbsp;inflowing air was filtered (high efficiency particulate air [HEPA] filter and electromagnetic filter); (ii)\u0026nbsp;only one person handled samples and the sample holder while a second person operated the measuring system; (iii) both persons wore personal clean room equipment; and (iv) the exposure of samples to air was kept to a minimum (about two minutes for each measurement).\u003c/p\u003e\n\u003ch2\u003eLaser Ablation \u0026ndash; Inductively Coupled Plasma \u0026ndash; Mass Spectrometry (LA-ICP-MS)\u003c/h2\u003e\n\u003cp\u003eDistributions of iron, copper and manganese in the brain tissue were measured using LA-ICP-MS. Data were recorded on one mesencephalon sample from each case (157/22, left hemisphere; other cases, right hemisphere). LA-ICP-MS measured the two-dimensional distribution of selected isotopes by ablating particles from the sample surface with a laser, which were directed to ICP-MS by an Argon gas flow and analyzed. To achieve plane sample surfaces required by this method, mesencephalon samples from each of the four brainstems were embedded in methylmethacrylate (MMA) according to existing protocols\u003csup\u003e38,39\u003c/sup\u003e. To this end, the samples were first dehydrated in ethanol baths of increasing concentration (70, 80, 90, 100%), then degreased in xylene, followed by incubation in 100% methanol. Finally, the samples were embedded in MMA (product number: 800590, Sigma Aldrich, St. Louis, MO, USA). To minimize contamination with magnetic particles, chemicals were filtered with PTFE filters (mesh size, 0.05 \u0026micro;m) except for more viscous chemicals that were filtered with a 0.2 \u0026micro;m mesh. The chemicals were filtered into glasses that were pre-washed with 10% HCl and rinsed with filtered distilled water. Slices of approximately 600 \u0026micro;m in thickness were cut from the MMA blocks using a circular saw microtome (SP 1600, Leica, Wetzlar, Germany). The slices were ground and polished with a 400 CS micro-grinder (EXAKT Advanced Technologies, Norderstedt, Germany), which decreased the slice thicknesses to approximately 400 \u0026micro;m; surfaces were thoroughly cleaned with isopropanol after polishing.\u003c/p\u003e\n\u003cp\u003eThe LA-ICP-MS instrument consisted of a laser ablation system (NWR-213, New Wave Research Inc., Fremont, CA, USA) coupled to a NexION 300 ICP-MS (PerkinElmer, Waltham, MA, USA), with distributions of two iron isotopes (\u003csup\u003e56\u003c/sup\u003eFe, \u003csup\u003e57\u003c/sup\u003eFe), copper (\u003csup\u003e63\u003c/sup\u003eCu) and manganese (\u003csup\u003e55\u003c/sup\u003eMn) being continuously recorded (Supplementary Table 3). For each sample, an area of 5 x 1.7 mm was selected covering parts of the Substantia nigra pars compacta (SNpc), which was easily visible due to large amounts of cells containing neuromelanin. Besides this being an area of interest for metals in the brain, the neuromelanin was used for orientation since the samples remained unstained for LA-ICP-MS to avoid possible contamination from staining. The scanned area was selected to extend from the SNpc to the red nucleus (RN) when possible. For case 159/22, the area was increased to 6 x 1.7 mm to scan a larger portion of the RN. The scans were oriented in a similar way for each sample, with the SNpc on the left side of the scanned area (i.e., during each line, the laser ablated first from the SNpc, then from white matter and lastly from the RN). Scans were done line-wise with a total of 67 lines per scan; intensities were uncalibrated. For each scan, data from the first ten seconds of each line were discarded due to the delay between laser ablation and ICP-MS measurement; data were analyzed using Laser Ablation App 1.0 (unpublished).\u003c/p\u003e\n\u003ch2\u003eLight Microscopical Analysis\u003c/h2\u003e\n\u003cp\u003eFollowing LA-ICP-MS, the brainstem sections were stained with Toluidine blue and imaged using a digital single-lens reflex camera (EOS 5D, Canon Inc., Ōta, Tokyo, Japan) equipped with a macro objective (LM macroscope 42x XL, Micro Tech Lab, Graz, Austria).\u003c/p\u003e\n\u003cp\u003eStained liver tissue samples were imaged with a light microscope (Axio imager.M2, ZEISS, Oberkochen, Germany) equipped with a 40x objective (Plan-Apochromat 40x, ZEISS, Germany) and operated with Stereo Investigator software (MBF Bioscience, Williston, VT, USA). Images were analyzed using an image browser (Biolucida Viewer, MBF Bioscience). Figures were constructed using a raster graphics editor (Affinity Photo 2, Serif Europe Ltd., Nottingham, UK). Only contrast and brightness adjustments were made with this editor, without altering the appearance of the original materials.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eData were analyzed with customized scripts in Python programming language (version 3.9, Python Software Foundation, Wilmington, DE, USA). Statistical tests included Mann-Whitney-U tests for independent samples and Wilcoxon signed-rank tests for dependent samples, both using confidence levels of 95%.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1.\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ward, R. J., Zucca, F. A., Duyn, J. H., Crichton, R. R. \u0026amp; Zecca, L. 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Mater.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e32,\u0026nbsp;\u003c/strong\u003e1042\u0026ndash;1051; 10.1016/j.dental.2016.05.012 (2016).\u003c/p\u003e\n\u003cp\u003e40.\u0026nbsp; \u0026nbsp;Kaub, L.\u003cem\u003e\u0026nbsp;et al.\u0026nbsp;\u003c/em\u003eMagnetic and LA-ICP-MS data for magnetic iron-oxide nanoparticles in the brain connected to alcohol-associated liver disease. \u003cem\u003efigshare\u003c/em\u003e \u003ca href=\"https://doi.org/10.6084/m9.figshare.25908568\"\u003ehttps://doi.org/10.6084/m9.figshare.25908568\u003c/a\u003e (2024).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank Hans-Georg Frank for helpful advice and Claudia Harbauer, Andrea Haderer, Beate Aschauer and Manuela Wei\u0026szlig; for skillful technical assistance. Laser Ablation App 1.0 was kindly provided by Daniel Hemmler. This work was supported by the German Research Foundation (DFG project GI712-25/1).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eL.K., S.M., C.S. and S.A.G. designed research; L.K., S.M., A.B., B.M. and S.A.G. performed research; L.K. and S.A.G. analyzed data; L.K., S.M., N.B., C.S. and S.A.G. interpreted results; and L.K., C.S. and S.A.G. wrote the paper and all authors read and contributed comments to the work.\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eAll source data are available on the figshare repository at \u003ca href=\"https://doi.org/10.6084/m9.figshare.25908568\"\u003ehttps://doi.org/10.6084/m9.figshare.25908568\u003c/a\u003e (ref.\u003csup\u003e40\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eCompeting Interests Statement\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"brain magnetite, brain iron, iron overload, LA-ICP-MS, alcohol use disorder","lastPublishedDoi":"10.21203/rs.3.rs-4731478/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4731478/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Magnetic iron-oxide nanoparticles in the form of magnetite (Fe3O4) are present in the human brain. They have been hypothesized to biomineralize in situ, as a result of dysfunctional iron homeostasis related to Alzheimer’s disease, or to enter the brain as airborne pollution particles. Regardless of their origin, magnetic iron-oxides pose a potential hazard to human health due to their high redox activity and surface charge. Here we report measurements on four post-mortem human brainstems, with one brainstem showing approximately 100 times higher magnetite concentrations than the other cases. This brainstem came from a subject with alcohol-associated liver disease (ALD) that manifested in liver cirrhosis and massive hepatic iron overload. Laser ablation – inductively coupled plasma – mass spectrometry showed the highest levels of trace metals (iron, copper and manganese) in the ALD brainstem. It is well established that a dysfunctional liver can result in the accumulation of trace metals in the brain. Our data indicate a similar pathway for magnetite particles, yet liver pathology has not been linked to magnetite occurrence in the brain so far. It may prove to be a crucial factor in understanding the high variation of magnetite concentrations found in human brains.","manuscriptTitle":"Magnetic iron-oxide nanoparticles in the brain connected to alcohol-associated liver disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-13 21:03:00","doi":"10.21203/rs.3.rs-4731478/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-25T06:06:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-20T17:03:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108757118530420655301626110140176342232","date":"2024-11-08T11:58:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-29T13:10:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271462568558495018497598995748197109384","date":"2024-10-17T09:55:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11344936556026544611584247509265982811","date":"2024-08-06T18:40:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-30T19:03:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-30T17:37:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-27T10:57:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-24T12:50:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-12T16:00:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d5c003c-d521-4ed0-9e53-8cbadd71ab12","owner":[],"postedDate":"August 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35680855,"name":"Health sciences/Gastroenterology/Hepatology/Liver diseases/Alcoholic liver disease"},{"id":35680856,"name":"Biological sciences/Biochemistry/Metals/Iron"},{"id":35680857,"name":"Physical sciences/Nanoscience and technology/Nanotoxicology/Medical toxicology"},{"id":35680858,"name":"Biological sciences/Neuroscience"},{"id":35680859,"name":"Physical sciences/Materials science/Nanoscale materials/Magnetic properties and materials"}],"tags":[],"updatedAt":"2025-07-14T16:00:15+00:00","versionOfRecord":{"articleIdentity":"rs-4731478","link":"https://doi.org/10.1038/s41598-025-09756-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-08 15:57:10","publishedOnDateReadable":"July 8th, 2025"},"versionCreatedAt":"2024-08-13 21:03:00","video":"","vorDoi":"10.1038/s41598-025-09756-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-09756-8","workflowStages":[]},"version":"v1","identity":"rs-4731478","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4731478","identity":"rs-4731478","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}