Brain Iron Accumulation in Neurodegenerative Disorders: Does Air Pollution Play a Role?

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Jithin V. George, Kathryn J. Hornburg, Alyssa Merrill, Elena Marvin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5314480/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 May, 2025 Read the published version in Particle and Fibre Toxicology → Version 1 posted 4 You are reading this latest preprint version Abstract Background Both excess brain Fe and air pollution (AP) exposures are associated with increased risk for multiple neurodegenerative disorders. Fe is a redox-active metal that is abundant in AP from traffic and industrial sources and in U.S. subway systems. Exposures to AP and associated contaminants, such as Fe, are lifelong and could therefore contribute to elevated brain Fe observed in neurodegenerative diseases, particularly via nasal olfactory uptake of ultrafine particle AP. These studies tested the hypotheses that exogenously generated Fe oxide nanoparticles could reach brain following inhalational exposures and produce neurotoxic effects consistent with neurodegenerative diseases and disorders in adult C57/Bl6J mice exposed by inhalation to Fe nanoparticles at a concentration similar to those found in underground subway systems (~ 150 ug/m 3 ) for 20 days. Olfactory bulb sections and exposure chamber TEM grids were analyzed for Fe speciation. Measures included brain volumetric and diffusivity changes, levels of striatal and cerebellar neurotransmitters and trans-sulfuration markers, quantification of frontal cortical and hippocampal Aβ42, total tau and phosphorylated tau and behavioral alterations in locomotor activity and memory. Results Particle speciation confirmed similarity of Fe oxides (mostly magnetite) found on chamber TEM grids and in olfactory bulb. Alzheimer’s disease (AD) like characteristics were seen in Fe-exposed females including increased olfactory bulb diffusivity, impaired memory and increased accumulation of total and phosphorylated tau, with total hippocampal tau levels significantly correlated with increased errors in the radial arm maze. Fe-exposed males showed increased volume of the substantia nigra pars compacta, a region critical to the motor impairments seen in Parkinson’s disease (PD), in conjunction with reduced volume of the trigeminal nerve and optic tract and chiasm. Conclusions Inhaled Fe oxide nanoparticles appeared to lead to olfactory bulb uptake. Further, these exposures reproduced characteristic features of neurodegenerative diseases in a sex-dependent manner, with females evidencing features similar to those seen in AD and effects in regions in males associated with PD. As such, prolonged inhaled Fe exposure via AP may be a risk factor for neurodegenerative diseases, and regulation of air Fe levels in enclosed areas like subway stations may have broad public health protective effects. More research is needed to improve our translation from these rodent studies to human exposures. We suggest, prolonged inhaled FE exposure via AP is a suggested risk factor for neurodegenerative changes in mice and given the similarities of these changes to changes observed in human AD, these data may have broad public health protections effects. iron air pollution tau olfactory bulb memory Alzheimer’s disease Parkinson’s disease substantia nigra Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 BACKGROUND Excess brain iron (Fe) accumulation is seen in numerous neurodegenerative diseases and disorders [ 1 , 2 ], and, given the redox activity of Fe, therefore often considered to play a key role in the etiology of these disorders [ 3 ]. In Alzheimer’s disease (AD), for example, a female-biased disorder characterized by the accumulation in brain of beta amyloids and tau and with dementia and cognitive decline, brain Fe levels are elevated above those associated with normal aging, even when concurrent elevations in serum Fe are not seen [ 4 , 5 , 6 ]. Cortical Fe levels have been found to correlate with the Braak staging of the severity of AD [ 7 ]. Similarly, Fe accumulation is found in substantia nigra and the red nucleus [ 8 ] even in early stages of Parkinson’s Disease (PD), a male-biased neurodegenerative disease resulting from the loss of dopamine neurons in substantia nigra that lead to motor impairments [ 9 , 10 , 11 ]. Further, elevated brain Fe has been shown to be negatively associated with general cognitive ability, and positively associated with cognitive aging in normal elderly individuals and even in children [ 12 , 13 ], and cognitive impairments are found in both AD and PD [ 14 , 15 , 16 , 17 , 18 ]. Despite the historical recognition of elevated brain Fe content in neurodegenerative diseases and disorders, the basis for this accumulation is not known, including in what forms excess Fe is incorporated at cellular levels. Excess brain Fe accumulation has typically been ascribed to e.g., dysregulation of Fe homeostasis [ 19 , 20 , 21 , 22 ] or breakdown of the blood brain barrier [ 23 ]. What has never been considered, however, is the hypothesis that exposure to Fe via air pollution (AP) may play a primary role, elevating exogenous Fe levels in brain, and promoting dysregulation of endogenous Fe homeostasis in response to uptake of exogeneous Fe-laden nanoparticles. Indeed, Fe constitutes one of the most abundant metal contaminants of AP [ 24 ], and exposures to AP are life-long. AP is a dynamic and complex mixture of gases (e.g., SO 2 and NO x ) and particulate matter (PM), with PM defined by particle size (i.e., coarse particles or PM 10 = ≤10 um, fine particles or PM 2.5 =≤2.5 um; ultrafine particles or UFP or PM 0.1 =≤100 nanometers) [ 25 ]. The UFP component is considered the most reactive component of AP given its greater surface area/mass ratio for adherence of contaminant particles that include metals such as Fe and other trace elements [ 26 , 27 ]. Of particular note UFPs, including metal oxides like Fe, are able to travel directly from deposits in the nose into the brain via olfactory axons and thereby bypass the blood brain barrier [ 28 , 29 ], which could explain the corresponding lack of any consistent elevations in serum Fe in AD and PD [ 30 , 31 , 32 ]. Further, mechanisms to remove excess Fe from brain are not apparent [ 33 ] and were likely not needed prior to industrialization. In accordance, AP exposures have been associated in epidemiological studies and in systematic reviews with multiple neurodegenerative diseases, including both AD and PD [ 34 , 35 , 36 , 37 , 38 ]. More specifically, exposures to the PM 2.5 (size fractions ≤2.5 um), component of AP as available through monitoring, have been associated with reduced memory and processing speed [ 39 , 40 ] and increased cognitive impairment [ 41 ], with some studies suggesting a more pronounced impact on cognitive decline in women [ 42 ]. Both PD and AD diagnoses has been correlated with levels of NO x [ 43 ], a marker of traffic density, and with levels of PM 2.5 [ 41 , 44 ]. It is important to note that UFP is a component of PM 2.5 . AP exposures have also been reported to alter brain structure, with reports of reductions in both white matter and gray matter volumes (reviewed in [ 45 , 46 ]) and in hippocampal volume [ 47 ] and with smaller deep-gray matter brain volumes and white matter volumes in frontal and temporal lobes, reductions in size of the corpus callosum, ventriculomegaly, and increases in brain infarcts [ 42 , 48 , 49 , 50 , 51 , 52 , 53 ]. Animal studies support these epidemiological associations of AP with neurodegenerative disease [ 46 ] and provide mechanistic links. A 15-week exposure to re-suspended urban PM 2.5 nanoparticles of wild type or EFAD mice that carry either ε3 or ε4 transgenes for human APOE, a gene known to enhance vulnerability to AD, in conjunction with five familial AD mutations, resulted in increased Aβ levels [ 41 ]. Additionally, both wild type and transgenic mice exhibited selective hippocampal neuronal atrophy. Exposures of wild type and TgF344-AD rats to real-world traffic-related AP resulted in AD neuropathology, including increased neuroinflammation and amyloid plaque deposition, higher levels of hyperphosphorylated tau, neuronal cell loss, and cognitive deficits in an age-, genotype-, and sex-dependent manner [ 54 ]. Real-world, concentrated UFP exposure resulted in alterations in memory domains [ 55 ], with altered microglial morphology and elevated phosphorylated tau in 3xTgAD mice [ 56 ]. In addition, exposures of aged rats for 3 months to PM 2.5 produced motor impairments and reduced tyrosine hydroxylase expression in olfactory and nigrostriatal circuits, increased radial diffusivity in MRI assessments and enhanced age-related demyelination [ 57 ]. In another study, an 8-week exposure to ambient fine or ultrafine particles reduced locomotion in rats in conjunction with decreased dopamine uptake [ 58 ]. Of the many metals/trace elements in AP, Fe is often one of the most abundant [ 24 , 59 , 60 ]. Fe levels in air measured at 13 sites in the U.S. averaged 108 ng/m 3 with a range of 41–240 ng/m 3 [ 61 ]. Furthermore, highly racially segregated counties sustain higher mass concentrations of fine particulate metals, indicating the importance of evaluating specific metal constituents of AP [ 62 ]. While collectively summed U.S.-wide ambient maximums indicate levels of 1210 µg/m 2 /day of Fe that include broad spatial variation based on emission sources [ 63 ], Fe levels can be concentrated in particular areas and reach extraordinarily high concentrations. For example, ambient PM exposures in underground U.S. subway systems were recently estimated to range from 112 ± 46.7 to 779 ± 249 µg/m 3 [ 64 , 65 ] and in other studies from 141 ± 81.1 to 329 ± 116 µg/m 3 [ 66 ]. While Fe in ambient AP from different sources can occur in water-soluble (Fe2 + ) forms [ 67 ], it can also be present as Fe (II, III) oxide: i.e., magnetite (Fe 3 O 4 ) and hematite (Fe 2 O 3 ) [ 68 , 69 , 70 , 71 , 72 ]. Analysis of Fe speciation is critical, as solubility and bioavailability of these metals influences redox potential that can produce oxidative stress. Magnetite nanoparticles, a mixed oxide with Fe 2+ /Fe 3+ , have been found in human postmortem brain samples [ 69 , 71 , 72 , 73 , 74 , 75 ], and brain magnetite has been linked to the incidence of AD [ 76 , 77 ] and found to be directly associated with both Aβ plaques and tau tangles [ 78 , 79 , 80 ]. That Fe-contaminated AP may be a source of excess brain Fe is suggested by two studies. In one, an abundant presence of magnetite (Fe 2+ /Fe 3+ iron oxide) nanoparticles approximately 10–150 nm in size, interpreted as being consistent with an exogenous rather than endogenous source of Fe formation based on crystal morphologies that pinpoint to high temperature formational mechanisms such as during coal combustion, was identified in frontal cortex of brains from AD patients [ 81 ]. Additionally, a recent report found an accumulation of ambient black carbon particles (a component of air pollution particulate matter) in thalamus, prefrontal cortex and olfactory bulb and hippocampus in post-mortem brains from individuals with neuropathologically confirmed Alzheimer’s disease [ 82 ]. Taken together, these data highlight the need to evaluate the neurotoxicity of inhaled Fe-oxide nanoparticles in AP and their potential relationship to neurodegenerative disorders such as AD and PD. For that purpose, the current study sought to confirm in a mouse model that: (i) brain uptake of exogenous Fe nanoparticles via inhalation exposure occurs, and (ii) to examine the hypothesis that inhalation of such Fe oxide nanoparticles could reproduce features of neurodegenerative diseases/disorders in a mouse model. The focus of the consequences of the exposures included some features unique to AD and PD, as well as some features shared across neurodegenerative diseases and disorders [ 83 ]. Sex-related differences in brain Fe metabolism as well as in accumulation with aging have been reported and may contribute to sex-bias in neurodegenerative disorders [ 84 , 85 ], and thus sex-related differences in outcome were also hypothesized. METHODS Animals and Husbandry : Male and female young adult (10 weeks old) C57Bl/6J mice from Jackson labs were placed in same-sex pairs and housed in standard mouse caging maintained at 22+-2 °C on a 12-h light-dark cycle (lights on at 06:00) at the University of Rochester Medical Center. Cages were provided with approximately 3 mm high performance bedding (BioFresh), ad libitum standard rodent chow (LabDiet Autoclavable Diet 5010) and water. Male and female mice were randomly assigned to either control or Fe-exposure groups. As shown in Fig. 1 , a subset of mice were euthanized at 48 hours post final Fe exposure and brains collected for Fe speciation analyses and magnetic resonance histological (MRH) analyses (n = 3/4 sex/treatment group) and for analyses of neurotransmitter levels (n = 6/sex/treatment group) in frontal cortex and cerebellum. An additional subset of mice were randomly selected for behavioral assessments and brains collected following euthanization at the termination of behavioral testing at approximately postnatal day 270, i.e., approximately 6 mos post-exposure, for evidence of protein aggregation (n = 12/sex/treatment group) and brain neurotransmitter levels. All mice used in the experiment were weighed every other day to monitor for signs of exposure-related systemic toxicity and treated humanely to alleviate suffering where possible in accordance with protocols approved by the Institutional Animal Care and Use Committees at the University of Rochester. For all mouse behavioral experiments and biochemical assessments, mouse/samples were counterbalanced by treatment group and sex to ensure any temporal or experimental variation was distributed across all groups. Fe Oxide: Electric Spark Generation and Exposure : Mice were exposed to Fe oxide via inhalation in compartmentalized whole body exposure chambers. For this study, the intended Fe concentration was 100 µg/m 3 which was chosen to be below the range of values cited for subway Fe levels. Exposures were carried out for 2 hours per day [ 86 ]. Mice were exposed for 5 days/week (M-F) over one month (July 8th, 2021 to August 6th, 2021) for a total of 20 exposure sessions. This exposure paradigm was roughly designed based on Organisation for Economic Co-Operation and Development (OECD) subacute inhalation toxicity study guidelines [ 87 ]. Whole-body inhalation exposures were conducted in the University of Rochester Inhalation Core Facility in single-house 30L stainless-steel reinforced Lexan exposure chambers. Control mice were exposed to HEPA-filtered air and experimental mice were exposed to Fe-oxide UFP particles generated by electric spark discharge in argon between two 99.99% pure iron rods (3N5 Purity, ESPI Metals, Ashland, OR, USA) using a GFG-1000 Palas generator (Palas GmbH, Karlshrue, Germany) in an argon atmosphere. Airborne particles were passed through a deionizer so that particles reached Boltzmann equilibrium charge. Particle number concentration was controlled by spark discharge frequency. Aerosol number concentration and particle size were monitored in real-time via a Condensation Particle Counter (CPC, model 3022, TSI Inc, St Paul, MN, USA) and Scanning Mobility Analyzer (SMPS, model 3934 TSI Inc, St Paul, MN, USA) respectively. The Fe-oxide particles (FeO, Fe 2 O 3 , and Fe 3 O 4 nanoparticles) were generated by adding a low flow of oxygen (~ 50 mL/min) into the argon flow (~ 5 L/min) which then entered the spark discharge chamber. An O 2 sensor (MAXO2 -250E, Maxtec, Salt Lake City, UT, USA) confirmed the maintenance of an oxygen concentration of 21% in the exposure chamber. This procedure produced particle sizes exclusively in the ultrafine size range with a count median diameter (CMD) of approximately 30–34 nm. Mass concentrations were determined gravimetrically by filter weight (25mm, Emfab Membrane Filters, Pall Life Sciences, Port Washington, NY) collected twice daily (5 L/min for 60 min., 300L total volume) from the filtered air and ultrafine Fe-oxide particle exposure chambers and secondarily determined using ICP-MS data. Electrostatic precipitation was used to collect particles on transmission electron microscopy (TEM) grids made of copper (add grid details). Magnetic Resonance Histology : Magnetic resonance histology (MRH) was performed using methods previously described fully [ 88 ], conducted similarly to magnetic resonance imaging only these evaluations are conducted post-mortem. Briefly, mice were perfusion-fixed using an active stain of buffered formalin and Prohance, a Gd contrast agent used to reduce the spin lattice relaxation time (T1) with imaging using a 9.4T vertical bore magnet [ 88 ]. These specimens use the same diffusion weighted imaging protocol that has been ported to a 7T horizontal bore magnet with similar, gradient, and rf coils. A 3D spin echo Steskal Tanner sequence was used with TR/TE = 100/15.8 ms with isotropic spatial resolution of 35 microns. Forty-six 3D volumes were acquired with b values of 3000 s/mm2 with b vectors that uniformly sample the unit sphere. Five baseline (b0) volumes were acquired. All data was acquired with compressed sampling using an acceleration of 8X and reconstructed using the iterative methods described in [ 89 ]. Labels were applied using the methods described in [ 88 ]. The labels (r1CCFv3) are consistent with the Allen Brain Atlas common coordinate framework with modifications to accommodate quantitative connectomics. The b0 volumes were registered together. The 46 diffusion weighted volumes were registered to this average baseline to reduce the consequences of eddy current. The resulting 4 dimensional volume was post processed in DSI Studio ( https://dsi-studio.labsolver.org/ ) yielding the following quantitative scalar images: axial diffusivity (AD), mean diffusivity (MD), radial diffusivity (RD), fractional anisotropy (FA), color fractional anisotropy (clrFA) all of which provide insight into the tissue cytoarchitecture [ 90 ]. Behavioral Assessment: Locomotor Activity – Spontaneous locomotor activity was measured in one 60-minute session in chambers (27.3 cm x 27.3 cm x 20.3 cm) that contained 48-channel infrared photobeams (Med Associates Inc., St. Albans, Vermont). Photobeam breaks were recorded across five-minute intervals for 60 minutes using measures of stereotypic, vertical, and ambulatory movements as well as ambulatory distance and time in center vs. edge zones. Stereotypic counts were defined as localized movement, i.e., the number of beam breaks within a 2x2 inch photobeam box when non-ambulatory. Vertical counts were defined as the total time that z-axis photobeams or photobeams that were 7 cm above the floor of the locomotor box were broken. Ambulatory counts were defined as the number of photobeam breaks during ambulatory movement, and ambulatory distance was defined as the differences in angular movements. Time in zone was defined as the total time spent within a given zone. Resting time was defined as time spent with no new photobeam breaks. Zone entries were defined as entry of all four paws into a given zone. The locomotor arena was broken into two zones: the center zone (center 15.7 x 15.7 cm square) or edge zone (space between center square and arena boundaries). Novel Object Recognition Memory (NOR ) – Following the locomotor activity session, NOR assessment was carried out. NOR consisted of two sessions conducted in an open plexiglass arena (30.5 cm x 30.5cm x 30.5cm). During the first session, mice were placed individually into the test chamber containing two small round white knobs secured to the chamber floor, and were allowed to explore the chamber and objects for 10 min. The purpose of this initial session was to allow mice to become familiar with the sample objects and for assessment of potential side preference and overall exploration patterns across exposure groups. The second session occurred 24 h after the first session to assess memory of the sample objects, premised on the ability of mice to detect and prefer novel stimuli. During the second session, mice were returned to the testing chamber, which now contained one small round, white knob (sample object) and one small square, black knob (novel object) in place of the prior white knob. Position of the novel object within the chamber (right or left side) was counterbalanced across treatments and subjects to preclude side bias. Both sessions were videotaped and scored using Noldus software (The Observer XT, Noldus) by a trained observer blinded to treatment condition. Object exploration was defined as a mouse being oriented toward the object with its head crossing a pre-marked 2 cm circle surrounding the object. Object recognition was analyzed using three different indices which control for differences in overall exploration across mice: duration index (total novel exploration time / [total novel time + total sample time]), bout index (total novel bouts / [total novel bouts + total sample bouts]), and time-per-bout index (average novel time per bout / [average novel time per bout + average sample time per bout]). Radial Arm Maze (RAM) – Following NOR testing, RAM performance was evaluated. The radial arm maze consisted of 8 arms emanating from a center arena. Mice were gradually food restricted until they reached 85% of their free-feeding body weight. Mice were then habituated to the radial arm maze in two sessions separated by 24 hours. In the first habituation session, two mice from the same cage were placed in the maze and allowed to freely explore. For the second habituation session, mealworms were placed at the end of each arm of the maze and then mice were individually introduced to the maze to freely explore for five minutes. Experimental sessions began 48 hours after the second habituation session. In subsequent experimental sessions, mealworms were placed in odd- or even-numbered arms, with placement counterbalanced by sex and treatment group. Mice were then placed in the center of the maze for five seconds with all arm doors closed. Then, doors were simultaneously raised and mice allowed to freely explore the maze. Arm entry was defined as all four paws of the mouse within an arm, past the arm door. If a mouse entered an arm, the door to the arm closed until the reward was completely consumed or for a total of five seconds. This process was repeated until all rewards were consumed or until the ten-minute maximum session time was reached, whichever occurred first. The maze was thoroughly cleaned with disinfectant between each test session. Male mice underwent 5 test sessions, while female mice underwent 3 test sessions due to disruptions by construction-related activity. Number of correct entries, number of incorrect entries or working memory errors (defined as repeat entries to arms after reward was already consumed), and time to obtain all rewards were recorded. Percent error was calculated as the ratio of number of incorrect entries to total number of entries multiplied by 100. Aβ42, Total and pS199 Tau Protein Quantification: At sacrifice (approximately postnatal day 270), hippocampus, frontal cortex, and striatum were dissected out and processed for ELISA analysis. Amyloid beta 42, total tau and pS199 tau concentrations were measured in duplicate using commercially available immunoassay kits (Invitrogen, Waltham, MA, USA, catalogue KMB3441, KMB7011 and KMB7041 respectively) according to the manufacturer’s specifications. Samples were read on a SynergyH1 Hybrid Reader (BioTek, Winooski, VT, USA) with Gn5 2.01 software. Sample replicates with coefficient of variation (COV) higher than 15% were excluded from the analysis. Standard curve CVs fell below 10%. Brain Neurotransmitter Levels Striatal and cerebellar concentrations of dopamine (DA), 3-4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), tyrosine (Tyr), norepinephrine (NE), glutamine (Gln), glutamate (Glu), gamma-aminobutyric acid (GABA), tryptophan (Trp), kynurenine (Kyn), serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) were measured in brains collected 48 hr post exposure to Fe and in another subset of mice at approximately PND270. For this purpose, hemisected brain tissue was thawed and diluted with 75 µL of ice-cold acetonitrile (50%, v/v) and homogenized via sonication for 10 seconds (SLPe digital sonifier, Branson Ultrasonics Corp., Danbury, CT.). Samples sat for ten minutes after which homogenates were collected and centrifuged at 10,000g for 20 mins and 4°C. The new supernatant was then collected and stored at -80°C until LC-MS analysis. Stock solutions of the above analytes were made at 5 mg/mL in ddH 2 O, except Tyr which was made in 0.2 M HCl. To study endogenous neurotransmitter variations within specific regions, standard solutions were made with ddH2O with analyte concentrations ranging as per prior range-finding studies. The solution was then derivatized using 13C6 benzoyl chloride (BzCl, Sigma Aldrich) as described by Wong et al., to create individual neurotransmitter internal standards. Internal standards were aliquoted and stored at -80°C until LC-MS analysis [ 91 ]. Right before analysis, internal standard mixtures were thawed, diluted in 50% acetonitrile and mixed with 1% sulfuric acid. This mixture was then added to derivatized samples. Samples were then centrifuged at 16,000g for five minutes and 20 µL of supernatant collected into a LoBind tube (Eppendorf). 10 µL of 100 mM sodium carbonate, 10 µL of 2% BzCl in acetonitrile, and 10 µL of internal standard were added sequentially to the LoBind tube. 50 µL of ddH2O was then added to reduce organic concentration and then, samples were centrifuged to remove any remaining protein pellets. The resulting supernatant was added to an autosampler vial. LC-MS/MS analysis was carried out by a Dionex Ultimate 3000 UHPLC coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher). Analytes were separated on a Waters Acquity HSS T3 column. The mobile phases were: 10 mM ammonium formate in 0.1% formic acid, and also, acetonitrile. The flow rate was set to 400 µL/min and the column oven was set at 27°C. After 5 µL of each sample was injected, the analytes were separated using a 12-minute multi-step gradient. The Q Exactive Plus was operated in positive mode, and a parallel reaction monitoring method (PRM) was used to detect derivatized molecules. Fragment ions were extracted with a 10 ppm mass error using the LC Quan node of the Xcalibur software (Thermo Fisher). Endogenous analyte peak areas were compared to those of each internal standard to determine relative abundance. These values were then divided by wet weight of the sample and then divided by air control to yield percent of control values. Turnover of neurotransmitters was also calculated including Gln/Glu, Glu/GABA, 5HIAA/5HT, HVA/DA and DOPAC/DA. Fe Nanoparticle Speciation in Brain: Distinguishing Exogenous and Endogenous Fe Fe nanoparticle speciation was identified in olfactory bulb (OB) thin sections using high-resolution scanning/transmission electron microscopy (S/TEM) coupled with spectroscopic elemental mapping. A JEOL 2100 F field emission S/TEM operated at 200 kV with analytic pole piece was used for the OB sections and also to identify the as-synthesized Fe-nanoparticles collected on TEM grids (Ted Pella, Inc. Redding, CA) for comparison. OB thin sections were obtained after brains were extracted and placed in filtered 4% PFA for initial tissue fixation. After dissection (using the right hemisphere), the OB tissues were post-fixed in 2.5% glutaraldehyde using 0.1M sodium phosphate buffer at 4°C followed by fixation in EPON-Araldite epoxy resin and then embedded in epoxy and polymerized at 60°C. All OB tissues were unstained to have a greater contrast of the Fe-nanoparticles with the cellular matrix. Tissue sections were cut to be ~ 70nm using an ultramicrotome (Boeckeler Instruments, Inc., Tuscon, AZ) and were placed onto nickel formvar/carbon coated slot grids (Ted Pella Inc., Redding, CA) to stabilize the tissue during beam interaction. High-resolution images of Fe nanoparticles in the OBs were recorded with a Gatan Ultrascan 4k CCD camera and data analysis and processing used Gatan Digital Micrograph software (Gatan, Inc.). The S/TEM analysis was coupled with spectroscopic elemental mapping of the Fe nanoparticles in the OB. A GATAN high angle annular dark field (HAADF) detector (Digiscan II) and an Oxfor Aztec EDS system from Oxford Instruments, Oxfordshire, United Kingdom were used. Energy dispersive spectroscopic analysis (EDS) was performed with a GATAN high angle annular dark field detector (HAADF), Digiscan II, Gatan 2000 Image Filter (GIF) with Oxford Aztec EDS software (Oxford Instruments, Oxfordshire, United Kingdom. All S/TEM images were acquired using an analytical probe with 0.17 nm. Statistical Analysis: Both male and female mice were used in all analyses. However, statistical analyses were stratified by sex based on known sex differences in response to Fe and of female bias in AD prevalence [ 1 , 92 , 93 , 94 , 95 , 96 ]. Brain neurotransmitter levels and levels of Aβ and tau were analyzed using one way analysis of variance (ANOVA). Locomotor activity data was analyzed in five-minute time intervals with repeated measures ANOVA; radial arm maze data was also analyzed by repeated measures ANOVA. NOR data were analyzed via one-way ANOVA separately for each session. Statistical analyses were conducted using JMP Pro 16.0 (SAS Institute Inc., Cary, NC, USA). P values ≤ 0.05 were considered statistically significant, while near significant values (p values ≤ 0.10) are also indicated. Outliers were first removed if determined by a statistically significant Grubb’s test (GraphPad Software Inc., San Diego, CA). Outlier analysis was never iterative, i.e., only one sample removed per treatment group per endpoint if they were confirmed by a Grubb’s test. For the diffusion MRI metrics and volumes, the statistical tests were again stratified by sex. Kruskal Wallis non-parametric ANOVA was used to determine the significance of change amount of the 180 x 2 regions within the atlas for the axial, radial, and mean diffusion contrasts and volume. For volume, the volume of each region was normalized by the total brain volume of each specimen prior to analysis. Secondly, an omnibus Kruskal Wallis test was performed with groups of Male Air versus Female Air versus Male Fe versus Female Fe to investigate whether the commonly significantly changed regions for the sex stratified changed in the combined model. A posthoc Dunn’s test with Sidak correction was used to identify the pairwise comparisons of groups that contributed most to any significant result. P values ≤ 0.05 were considered statistically significant. Eta 2 and Cohen’s F were used to quantify strength of the effect size for significant results. The statical calculations of MRI data were done in MATLAB. Prior to analysis of the MRI metrics and volumes, the values of the left and right structures were combined by summing volumes and performing the weighted mean on diffusion metrics. Parent structures are generated from all potential parents in the RCCF atlas. The volume of each region was normalized by the total brain volume. First, a 2-way ANOVA was carried out with interactions considering sex and iron exposure conditions. This was followed by a Kruskal-Wallis non-parametric ANOVA to consider each sex independently for the iron exposure condition. P values ≤ 0.05 were considered statistically significant. Eta 2 and Cohen’s F [ 97 ] were used to quantify strength of the effect size for significant results. Using G* Power [ 98 ], we estimated the minimal Cohen’s F effect size needed to maintain statistical power of 0.8. In the 2-Way ANOVA with interaction testing, the estimated Cohen’s F effect size needed to maintain sufficient power is 0.82, while for the stratified by sex Kruskal- Wallis Non-Parametric ANOVA, an effect size of 1.3 is needed to maintain the same power criteria. The statistical calculations of MRI data were done in MATLAB. RESULTS Fe Exposure Concentrations Mass concentration averaged 109.5 ± 24.16 ug/m 3 across the exposures, and mean particle counts averaged 2.01E + 06 #/cm 3 across the 20 exposure sessions ( Fig. 2 A ) . The particle diameter remained in the nanoparticle range (43–52 nm) across the course of exposure (Fig. 2 B). To compare deposition fractions in mice, a multiple-path particle dosimetry model (MPPD; Applied Research Associates, Inc. v3.04) was used, with settings customized to B6C3F1 mouse with subject-specific functional residual capacity (FRC), upper respiratory tract (URT), respiratory rate (RR), and tidal volume (TV). Additionally, density of iron-oxides was set to an average 5.2 g/cm 3 , based on the approximate density of singlet iron oxides (averaged between Fe3O4 and Fe2O3), with a median mass aerodynamic diameter (MMAD) of 0.04812 um as reported above. Using these parameters, estimates of particle deposition were modeled for comparison. The total modeled deposition fraction was 53.84%% with 33.48% depositing in the head, 15.61% depositing in the tracheobronchial region, and 4.7% depositing in the pulmonary region. Of the modeled pulmonary deposition, 16.53% deposited in the central respiratory airway and 3.83% deposited in the peripheral conducting respiratory airways. Fe Characterization, Speciation and Translocation to Olfactory Bulb Nasal olfactory uptake of Fe nanoparticles and its speciation was assessed in olfactory bulb, the port of entry into brain and compared to Fe speciation on TEM grids from the inhalation chamber. The STEM analyses with corresponding elemental maps and electron diffraction of the as-synthesized Fe particles are summarized in Fig. 3 and provide structural and compositional details for the grid collected Fe speciation from the exposure chamber which formed predominantly magnetite particles, i.e., Fe 3 O 4 . Most particles were spherical with polycrystalline domains although some euhedral crystals were recognized (Fig. 3 A a ). Elemental mapping for Fe and O is shown in Fig. 3 A b-c which indicated that oxygen is evenly distributed through the bulk of the particles. At higher resolution, the Fe 3 O 4 spheres have a narrow band at the surface (Fig. 3 A d-e ) that consists of hematite Fe 2 O 3 , a more oxidized Fe speciation that was further distinguished from the Fe 3 O 4 -rich core region of the Fe particles using electron diffraction (Fig. 3 A f-g ). The magnetite particles were also identified in the olfactory bulb after inhalation exposure and examples shown in Fig. 3 B a-m . The olfactory bulb has magnetite present in close proximity to neurons and astrocytes and also next to corpora amylacea which can be distinguished in the OB tissue regions using STEM imaging even in the unstained sections (Fig. 3 B a ). Two examples of translocated Fe particles are indicated in Fig. 3 B a and the larger Fe agglomerate is magnified in Fig. 3 B b-e with corresponding elemental maps for Fe and O. Results indicate that the magnetite (Fe 3 O 4 ) particles that translocated to the OB have the same chemical and crystalline structure as the spark-generated particles from the TEM grids. The surface rim of hematite (Fe 2 O 3 ) around the magnetite core that was identified in the TEM grids sample is missing in the olfactory bulb Fe particles which are characterized by a rougher surface layer potentially due to particle-tissue interaction and some bioprocessing of the particles that may cause partial dissolution along the surfaces (Fig. 3 B c-e ). Many of the Fe particles in the OB were not agglomerated or had only one or two other particles nearby, as shown in Fig. 3 B f-I with corresponding elemental maps for Fe and O. Near the exogenous Fe particles were also copious amounts of biomineralized iron in the form of ferritin nanoparticles which typically are 3–12 nm in diameter and do not agglomerate. The ferritin are typically smaller in size compared with the exogenous Fe (Fig. 3 B f; j, l-m ) and were identified using electron energy loss spectroscopy (EELS) analysis based on their FeL3 and FeL2 edges (Fig. 3 B d ). Thus, Fe 3 O 4 (magnetite) was detected on the grid post-exposure and Fe 3 O 4 particles were likewise found in olfactory bulb in an Fe-exposed brain. Fe nanoparticles are identified in the olfactory bulb Bottom “B”: STEM image shows two regions marked with yellow square where Fe particles translocated to the olfactory bulb (a). A magnified view of the agglomerate in (a) is shown in (b) and further magnified in (c)-(e) with corresponding elemental maps for Fe and O distribution. The STEM image in (f) illustrates the location of two isolated exogenous magnetite (Fe 3 O 4 ) particles near a corpora amylacea body with copious endogenous Fe particle accumulation “ferritin NP”. The Fe 3 O 4 are further magnified in (g)-(i) and show no Fe 2 O 3 rims. The ferritin NP are illustrated at higher magnification in the STEM images in (j) and (l) with corresponding elemental map for Fe in (m). ELLS analysis of ferritin NP of the region marked in (j) with a yellow square. Correspondingly, Fe 3 O 4 was detected in olfactory bulb from an Fe-exposed brain. Brain Neurotransmitter Levels Two Days Post-Exposure: Levels of glutamatergic, serotonergic and dopaminergic classes of neurotransmitters were examined in the striatum and cerebellum two days post termination of exposure. Striatum – Striatal levels of neurotransmitters in Fe- and air-exposed females (top row) and males (bottom row) are shown in Fig. 4 . No significant differences in levels of any of the neurotransmitters within any of the three classes examined were found in response to inhaled Fe in males. In the case of females, however, Fe-based changes were found in both glutamatergic and dopaminergic systems. Specifically, within the glutamatergic system, significant Fe-related increases were found in levels of glutamine (Gln: +19%, F(1,4) = 7.88, p = 0.048) and glutamate (Glu: +29%, F(1,4) = 15.38, p = 0.0172), with a marginal increase in levels of GABA (+ 31%, F(1,4) = 4.66, p = 0.097). Marginal Fe-related increases were observed in levels of the dopamine metabolites DOPAC (+ 118%; F(1,4) = 6.12, p = 0.069) and HVA (+ 83%; F(1,4) = 6.65, p = 0.061). Cerebellum - As in striatum, no significant changes were found in Fe-exposed male cerebellum in glutamatergic, dopaminergic or serotonergic neurotransmitter levels (Fig. 5 ). In female Fe-exposed cerebellum, significant increases were seen in levels of serotonin (5HT: +83%, (F(1,4) = 7.83, p = 0.049) along with marginal increases in the 5HT metabolite 5-HIAA (+ 97%, (F(1,4) = 7.47, p = 0.052) within the serotonergic system. Magnetic Resonance Histological Imaging Two Days Post-Exposure: Magnetic resonance histological imaging of brains collected within 48 hr of exposure revealed significant changes in mice exposed to Fe. Sex + Exposure + Sex:Exposure Model Table 1 shows the results of a 2-Way ANOVA with interaction model for diffusion tensor imaging, fractional anisotropy and volumetric changes. This gives a high-level overview of changes. Diffusion Tensor Imaging Changes – As shown in Table 1 , the olfactory bulb was significantly changed in response to Fe, with mean, radial and axial diffusivity contrasts for the sex:exposure interaction. The male and female response was in the opposite directions, with increases in Fe-exposed females in mean, radial and axial diffusivity, and reductions in mean, radial and axial diffusivity in olfactory bulb in Fe-exposed males. Post hoc testing (Tukey-Kramer HSD method via multcompare in MATLAB) indicated that for mean, radial and axial diffusivity, the female Fe versus male Fe was the most significant pairwise comparison set (p < 0.1). Table 1 Summary of MRI Changes with model of 2-Way ANOVA (Sex and Exposure), Interactions DIFFUSION TENSOR IMAGING CHANGES Structure Statistical Summary, Sex:Exposure MALE Air vs Fe FEMALE Air vs Fe Olfactory Bulb Mean: p < 0.01, Cohen’s F 1.04 Radial: p < 0.01, Cohen’s F 1.06 Axial: p < 0.01, Cohen’s F 0.982 Mean: -5.3% Radial: -4.9% Axial: -5.9% Mean: 5.0% Radial: 4.8% Axial: 5.3% FRACTIONAL ANISOTROPY CHANGES Structure Statistical Summary, Sex:Exposure MALE Air vs Fe FEMALE Air vs Fe Posterior Amygdalar Nucleus p < 0.05, Cohen’s F 0.847 -11.3% 5.2% VOLUMETRIC CHANGES Structure Statistical Summary, Sex:Exposure MALE Air vs Fe FEMALE Air vs Fe Geniculate Group, Dorsal Thalamus p < 0.01, Cohen’s F 0.948 3.5% -2.1% Medial Geniculate Complex, Ventral Part p < 0.05, Cohen’s F 0.919 5.1% -6.2% Medial Geniculate Complex p < 0.05, Cohen’s F 0.851 2.7% -3.9% P-values included in table are uncorrected. Negative (-) % change signifies that Air is larger and positive (+) % change signifies that Fe is larger. Volumetric findings were controlled by brain volume of each specimen, thus normalized volume. Sex is a significant for volumetric changes in the 2-Way ANOVA Models for these structures: Central Amygdalar Nucleus (Uncorrected p < 0.01, Cohen’s F 1.37), Anteroventral Nucleus of Thalamus, (Uncorrected p < < 0.01, Cohen’s F 1.07), Prelimbic Area (Uncorrected p < 0.01, Cohen’s F 0.876), Orbital Area (Uncorrected p < 0.05, Cohen’s F 0.888), Superior Colliculus-Sensory Related (Uncorrected p < 0.05, Cohen’s F 0.887), and Ventral Posteromedial Nucleus of the Thalamus (Uncorrected p < 0.05 Cohen’s F 1.00). There were no structures with significant changes in diffusion tensor metrics or normalized volume related to exposure alone. Fractional Anisotropy – Analyses revealed one structure, Posterior Amygdalar Nucleus, that was significantly changed for the sex:exposure interaction. Male mice exhibited a decrease in the fractional anisotropy following Fe exposure (-11.3%), while female mice showed an increase in response to Fe exposure (+ 5.2%). Volume Changes – There were significant changes in multiple components of the geniculate complex (most child to parent structure ordering: Medial Geniculate Complex, Ventral Part; Medial Geniculate Complex; Geniculate Group, Dorsal Thalamus). Specifically, male mice had increases in the volume (+ 5.1%, + 2.7%, and + 3.5%) with Fe exposure, while female mice showed decreases in volume (-6.2%, -3.9%, -2.1%) in response to Fe exposure. Stratified by Sex, Changes due to Exposure Table 2 shows the results of stratified by sex Kruskal Wallis non-parametric ANOVAs indicating significant diffusion tensor imaging, fractional anisotropy and volumetric changes. Diffusion Tensor Imaging Changes - Olfactory bulb was significantly influenced by Fe in both female and male mice, but again in opposite directions (Fig. 6 ). Additional analyses revealed sex-dependent changes in olfactory bulb diffusivity consistent with increased myelin damage and axonal loss. Specifically, the mean male response to Fe exposure (axial: 0.348 +/- 0.0171 10 − 3 x mm 2 /s, radial: 0.270 +/- 0.0105 10 − 3 x mm 2 /s, mean: 0.296 +/- 0.0127 10 − 3 x mm 2 /s) was lower than that than of the mean female response to Fe exposure (axial: 0.373 +/- 0.00649 10 − 3 x mm 2 /s, radial: 0.286 +/- 0.0.0360 10 − 3 x mm 2 /s, mean: 0.315 +/- 0.00441 10 − 3 x mm 2 /s), assisting in explaining why in the post hoc analysis of the overall 2-Way ANOVA model, this pairwise comparison was also the most changed, i.e., the most different pairing. Additional significant changes in the diffusion tensor metrics were unique to each sex. For male mice, there are significant reductions in olfactory areas (mean: -4.2%, axial − 4.8%), and hippocampal commissures (radial: -4.4%). For female mice, there are significant increases in the anterior olfactory nucleus (axial: +5.4%). Fractional Anisotropy – Alterations in fractional anisotropy in the MRI assessment were male-specific ( Table 2 ) . Fe-exposed males showed significant reductions in fractional anisotrophy (FA) in posterior amygdalar nucleus (-11.3%), hypoglossal nucleus (-10.7%), pretectal region (-10.3%), basal amygdalar nucleus (-7.0%), subicular regiona (-6.6%), cortical amygdalar zones (-6.4%), fimbria (-6.1%), cranial nerves (-5.6%), trigeminal nerve (-5.4%), and CA1 (-2.4%). Increases in FA in Fe-exposed males were seen in medial preoptic nucleus (+ 8.9%), nucleus accumbens (+ 7.9%), and ventral part of striatum (+ 4.1%). Volumetric Changes - As shown in Table 2 , male Fe-exposed mice showed a significant increase in the normalized volume of the substantia nigra compact part (+ 8.0%), midline group of the dorsal thalamus (+ 5.2%), the posterior complex of the thalamus (+ 4.1%), the spinal vestibular nucleus (+ 3.6%), and the geniculate group, dorsal thalamus (+ 3.5%). Males concurrently exhibited significant volumetric reductions in the trigeminal nerve (-6.0%), the optic tract and chiasm (-4.7%), epithalamus (-3.0%), and of the bed nuclei of the stria terminalis (-2.1%). Females showed a significant 5.5% increase in the normalized volume of the vestibulocerebellar nucleus with corresponding significant reductions in volume which were largest in the ventral medial geniculate complex (-6.2%, Table 2 ), but also showed significant reductions in the lateral amygdala nucleus (-3.6%) and the sensory related portions of the medulla (-2.4%). Table 2 Summary of MRI Changes with model of Kruskal Wallis non-parametric ANOVA on Exposure, Stratified by Sex DIFFUSION TENSOR IMAGING CHANGES MALE FEMALE Structure Air vs Fe Structure Air vs Fe + Olfactory Bulb Mean: -5.3% Radial: -4.9% Axial: -5.9% + Olfactory Bulb Mean: 5.0% Radial: 4.8% Axial: 5.3% Olfactory Areas Mean: -4.2% Axial: -4.8% Anterior Olfactory Nucleus Axial: 5.4% Hippocampal commissures Radial: -4.4% FRACTIONAL ANISOTROPY CHANGES MALE FEMALE Structure Air vs Fe Structure Air vs Fe + Posterior Amygdalar Nucleus -11.3% Hypoglossal Nucleus -10.7% Pretectal Region -10.3% Medial Preoptic Nucleus 8.9% Nucleus Accumbens 7.9% Basomedial Amygdalar Nucelus -7.0% Subicular Region -6.6% Cortical Amygdalar Zones -6.4% Fimbria -6.1% Cranial Nerves -5.6% Trigeminal Nerve -5.4% Striatum Ventral Region 4.1% CA1 -2.4% VOLUMETRIC CHANGES MALE FEMALE Structure Air vs Fe Structure Air vs Fe Substantia Nigra Compact Part 8.0% + Medial Geniculate Complex, Ventral Part -6.2% Trigeminal Nerve -6.0% Vestibulocerebellar Nucleus 5.5% Midline Group of the Dorsal Thalamus 5.2% Lateral Amygdalar Nucleus -3.6% Optic Tract and Chiasm -4.7% Medulla Sensory Related -2.4% Posterior Complex of the Thalamus 4.1% Spinal Vestibular Nucleus 3.6% + Geniculate group, dorsal thalamus 3.5% Epithalamus -3.0% Bed nuclei of the stria terminalis -2.1% + Structures which appeared significant in the 2-Way ANOVA analysis, considering sex and exposure together. For all entries, uncorrected P-values is < 0.05 and estimated Cohen’s F is 1.73. Negative (-) % change signifies that Air is larger and positive (+) % change signifies that Fe is larger. Volumetric findings were controlled by brain volume of each specimen, thus is normalized volume Aβ42 and Tau 6 Months Post-Exposure: As measured approximately 6 months post exposure to inhaled Fe, neither protein levels of pS199 tau, total tau, and Aβ42 concentrations in the frontal cortex or in hippocampus were affected in male mice (Fig. 7 ). In contrast, Fe-exposed female mice exhibited significantly higher concentrations in frontal cortex of pS199 tau ( t = 2.76, p = 0.02) and of total tau ( t = 3.63, p = 0.008), but similar concentrations of Aβ42 ( t = 0.99, p = 0.35) as compared to air-exposed controls. Fe-exposed female mice also exhibited significantly higher hippocampal concentrations of total tau ( t = 2.57, p = 0.028) than did air-exposed controls, but showed similar concentrations of hippocampal pS199 tau ( t = 1.50, p = 0.17) and Aβ42 ( t = -1.23, p = 0.25). Behavioral Changes Post Exposure: Locomotor Activity Levels - Fe-exposed males showed no significant differences from air-exposed males in any measures of locomotor activity levels: ambulatory distance, ambulatory episode, ambulatory time, jump counts, jump time, rest time, stereotypic counts, stereotypic time, vertical counts, or vertical time ( Supplementary Fig. 1 ). Similarly, females, regardless of treatment group, performed equivalently across all measures of locomotor behavior. Novel Object Recognition - In session 1 of NOR, mice, regardless of sex and treatment, spent equivalent amounts of time with both the left- and the right-placed object as determined by comparing time spent with left or right object vs. half of total interaction time (Fig. 8 ), confirming an absence of spatial or activity level bias that could influence results in session 2 determination of the NOR recognition index. Recognition index in session 2, calculated as time spent with the novel object divided by total time spent with both objects averaged 58% and 68%, respectively, for male and female air control mice, consistent with recognition of a novel stimulus. This recognition index was not influenced by Fe exposure in male mice. However, Fe-exposed female mice displayed a 31% significantly lower recognition index than their air-exposed controls ( t = -5.19, p = 0.0007). Radial Arm Maze - Radial arm maze performance was assessed over 3 sessions in females and 5 sessions in males (Fig. 9 ). No significant effects of Fe on percent errors were seen in male mice over the course of the 5 sessions of testing, with both groups showing chance levels of accuracy in session 1 and slight declines thereafter. In contrast, while chance levels of errors were also seen in female mice in sessions 1 and 2, levels of errors in female control mice dropped in session 3 by almost 30%, whereas no such change was found in Fe-exposed females, resulting in a marginally significant interaction in the repeated measures analysis (time x treatment (F(2, 9) = 0.060), with a significant day 3 reduction confirmed in a subsequent post-hoc t-test ( t = 2.86, p = 0.017). Notably, levels of percent errors in Session 3 in females (Fig. 9 , bottom ), including both air- and Fe-exposed mice, were significantly correlated with hippocampal levels of phosphorylated tau (r 2 = 0.37; F (1,11) = 6.44, p = 0.0275). Post Behavior Neurotransmitter Changes: Changes in striatal and cerebellar neurotransmitter levels were examined post behavioral testing in mice that underwent behavioral assessments. Striatum – In contrast to effects seen 48 hr post exposure, significant Fe-induced changes in striatal neurotransmitters post behavioral testing were seen only in males (Fig. 10 ). In Fe-exposed females, the only change seen was a marginal (8%) increase in excitotoxicity (glutamate/GABA; F(1,10) = 3.29, p = 0.0998). In contrast, changes in all three classes of neurotransmitters were now evident in males. Effects within the class of glutamatergic neurotransmitters included a significant 19% increase in levels of GABA (F(1,10) = 6.25, p = 0.031), a 40% marginal increase in levels of glutamine (F(1,10) = 4.59, p = 0.058) and a 22% marginal increase in glutamate (F(1,10) = 4.4, p = 0.062). Changes seen within the class of serotonergic neurotransmitters in Fe-exposed male striatum included an 84% marginal increase in kynurenine (F(1,10) = 4.71, p = 0.0552), as well as a 32% significant reduction in levels of serotonin (F(1,10) = 18.32, p = 0.0016). Change also occurred in response to Fe within the class of dopaminergic neurotransmitters, specifically in reduced dopamine turnover, with a 44% significant reduction in the ratio of HVA/DA (F(1,10) = 5.2, p = 0.046) as well as a significant 25% reduction in the ratio of DOPAC/DA (F(1,10) = 5.4, p = 0.043). Cerebellum - Post behavior changes in cerebellar neurotransmitter function (Fig. 11 ) were more limited. In females, Fe-induced changes were limited to a marginal 17% increase in levels of glutathione (F(1,12) = 4.1, p = 0.066). In males exposed to Fe, a significant 28% increase in levels of serotonin turnover were observed (F(1,12) = 5.69, p = 0.035) in conjunction with a marginal 20% increase in dopamine turnover (DOPAC/DA; F(1,12) = 3.52, p = 0.085). Trans-Sulfuration Markers Changes in levels of markers within the trans-sulfuration pathway, specifically methionine, homo-cysteine, cysteine and glutathione, were measured in both striatum and cerebellum at 2 days post exposure (labeled Pre) and after behavioral testing (labeled Post) and are depicted in Fig. 12 . In striatum, females evidenced significant increases in glutathione even at 2 days post Fe exposure (GSH: +23%, F(1,4) = 10.17, p = 0.0332), with levels remaining elevated albeit not significantly when measured post behavioral testing. Notably, males showed a marked 44% significant increase in glutathione (F(1,10) = 6.65, p = 0.028) but this was not evident until post behavioral testing, and was accompanied by a significant 31% increase in levels of cysteine (F(1,10) = 10.07, p = 0.0099). Some evidence of a delayed increase in glutathione was also seen in females in cerebellum (F(1,12) = 4.098, p = 0.066). DISCUSSION Based on the accumulating evidence linking both AP exposure [ 34 , 35 ] and elevated brain Fe [ 1 , 2 ] concentrations with risk for neurodegenerative diseases and disorders, the current study sought to examine in a mouse model the hypothesis that inhaled Fe, as would occur through AP exposures, would reach brain and would reproduce features of such diseases and disorders, and do so in a sex-dependent manner. Consistent with these hypotheses, speciation analyses of TEM grids from the exposure chambers confirmed the presence of spark-generated, exogenous magnetite which was likewise seen in olfactory bulb, the region that would be the initial port of entry into brain following nasal olfactory uptake in response to Fe inhalation, confirming the uptake of exogenous Fe. Additionally, characteristics of neurodegenerative diseases and disorders occurred in response to Fe inhalation and differed notably by sex. Specifically, in females, numerous outcomes characteristics of AD were seen, including increased levels of phosphorylated tau in frontal cortex and total tau in both frontal cortex and hippocampus, and increases in olfactory bulb diffusivity potentially indicative of myelin damage and/or axonal loss. Fe-exposed females also exhibited impaired memory, as assessed using both using a novel object recognition paradigm and a radial arm maze paradigm, with the latter impairments significantly correlated with levels of frontal cortical total tau. In contrast, the profile of consequences in males differed notably and showed characteristics associated with PD that included increases in volume of the substantia nigra pars compacta concurrently with reductions in the volume of the trigeminal nerve, in addition to reductions in mean, radial and axial diffusivity in olfactory bulb and hippocampus and altered fractional anisotropy changes in multiple subcortical structures. S/TEM analysis coupled with EDS confirmed the presence of magnetite in mouse olfactory bulb following Fe inhalation. These exogenous Fe nanoparticles can be identified based on their structural and crystalline similarity to the spark generated Fe nanoparticles produced and collected on TEM grids (Fig. 3 ). Such findings are consistent with nasal olfactory uptake of elemental AP particles and salts in both rats and humans [ 99 ], including Fe, Mn, Cd, Ni, Hg, Al, Co, Zn, and Cu [99, 100, 101, 102, 103, 104], through translocation across olfactory epithelium by olfactory neuronal cells along neuronal tracts, followed by transportation into olfactory bulb, and movement to other brain regions [ 105 ]. Sensory nerves in the upper and lower respiratory tract can also translocate particles [ 106 ] that reach e.g., the trigeminal ganglion or the vagal nerve [ 28 ]. Such Increases in Fe have been reported in brain and nerves of mice in other studies after inhalation exposures of Fe nanoparticles [ 107 ]. The olfactory route of exposure/uptake of Fe is of particular interest to reports defining the staging of neurodegenerative disorders, including AD and PD, as olfactory bulb is found as an early site of change in both [ 17 , 108 ]. In the current study, Fe-based changes in olfactory bulb diffusivity were found in both sexes, and appeared as reductions in diffusivity in males, but as increases in diffusivity in females. While it is not yet clear how such changes translate into neuropathological features, such findings could suggest potential myelin abnormalities and/or axonal changes [ 109 ]. Interestingly, in our prior studies of developmental air pollution exposures, hypermyelination of the corpus callosum occurred in both sexes following gestational exposures that was correlated with Fe inclusions in corpus callosum in females and axonal changes including thick myelin sheaths with “holes” indicative of damage [ 110 , 111 ], while postnatal exposures resulted in a male-biased hypomyelination [ 112 ]. Future research is needed to associate these AP related myelin changes with MR diffusivity measures. In terms of disease staging, Braak staging of PD suggests a pathogen entering the brain via the nasal route or trigeminal or via the vagal nerve as initial sites of pathology [ 17 , 113 ], consistent with nanoparticle routes to brain, with studies reporting olfactory bulb as an area that accumulates alpha-synuclein aggregates, a hallmark of PD. Of additional support is a study that examined metal concentrations and distributions in the human olfactory bulb in PD and found elevated Fe in the PD olfactory bulb [ 114 ]. In the case of AD, the unfolded protein response that leads to upregulation of beta-amyloid and tau production, hallmark features of AD, is found throughout the olfactory system even in early-stage Braak pathological staging of AD, including Braak stages 0 and 1 [ 115 , 116 ]. The olfactory bulb pathology indicated by increased diffusivity in MRI analyses in female Fe-exposed mice is consistent with the well documented involvement of olfactory bulb in dementia and AD, and indicative of changes to white matter microstructure. In accord with these findings, olfactory loss is considered to be a component of the long prodromal phase of AD as well as in PD [ 117 ]. Females showed several additional characteristic features of AD following Fe inhalation, including volumetric reductions in regions likewise implicated in AD and include structural connections with olfactory system [ 118 ]. For example, reductions in amygdala volume have long been recognized in AD [ 119 ] and have been found to be of greater magnitude in lateral amygdala [ 120 ], the specific nucleus in which volumetric reductions were seen in Fe-exposed female brain in this study. It is notable that hippocampus and amygdala show early involvement in AD and receive projections from the olfactory bulb [ 121 ]. In contrast to these regions, increases in size of the vestibulocerebellar nucleus (flocculus) were concurrently found in female Fe-exposed brains. While prior studies have cited involvement of cerebellum in AD, these were seen as reductions [ 122 , 123 ] as determined in individuals with diagnosed dementia. One potential basis for this increase could be an early compensatory plasticity. While compensatory mechanisms in AD have been proposed [ 124 ], the earliest changes in AD and possible compensatory processes have yet to be established. As with some other outcomes, studies of the time course of the consequences of Fe would provide important mechanistic information. Females exposed to Fe likewise showed evidence of impaired memory, with these impairments correlated with increased levels of phosphorylated tau in hippocampus. A hallmark of AD is the neuropathological misfolding and aggregation of two brain proteins, amyloid β (Aβ) and tau. Correlations between elevation of brain Fe and formation of neurofibrillary tangles have been reported [ 125 ]. Fe interacts with tau: Fe 3+ can induce aggregation of hyperphosphorylated tau, while reduction of Fe 3+ to Fe 2+ reverses the effect, as shown in hyperphosphorylated tau obtained from AD brains [ 125 , 126 ]. The reduction of Fe 3+ to Fe 2+ can solubilize iron since Fe 2+ can be present and transported in ionic form and hence be mobilized. Another way to reduce Fe 3+ to Fe 2+ is to reduce the common Fe 2 O 3 (hematite) to Fe 3 O 4 (magnetite) which does not mobilize or remove Fe from the tissue regions. The correlation of hippocampal phosphorylated tau levels with impaired memory is of particular interest given the reported role of tau in cognitive deficits in AD [ 127 , 128 ]. It has been suggested that entorhinal cortex tau accumulation underlies hippocampal activation and memory loss over time [ 129 ], while cerebrospinal levels of tau have been found to be predictive of reductions in hippocampal volume and interpreted as reflecting neuronal loss [ 130 ]. In AD, significant amounts of Fe are found within Aβ plaques and tau-based neurofibrillary proteins [ 131 , 132 , 133 ]. Although Fe promotes Aβ aggregation, and binds to the Aβ peptide with binding affinity increasing Aβ aggregation, further potentiating Aβ neurotoxicity [ 134 , 135 , 136 , 137 ], changes in Aβ were not found in these studies. It is possible that Aβ pathology needs to be evaluated in a transgenic mouse line with relevance to human AD protein production or requires more protracted exposures. A distinct effect found in males included a significant increase in volume of the substantia nigra pars compacta, the site of dopamine cell loss leading to motor dysfunction in male-dominated PD. The increase seen in substantia nigra volume is notable, given reports of enlarged substantia nigra hyperechogenicity that have been shown to correlate with Fe accumulation in substantia nigra [ 138 ], and constitute a predictive risk for PD [ 139 ], a male-biased disorder resulting from loss of dopamine neuronal cell bodies in this region. Notably, reductions in striatal dopamine turnover were observed concurrently. The increase in substantia nigra pars compacta volume seen here in males, again, could reflect an early compensatory response to Fe inhalation [ 140 ] and emphasizes the need to further understand compensatory responses particularly during the early trajectory of this disease. Additional studies are also needed to assess the relationship of hyperechogenicity to changes assessed by neuroimaging, as efforts to date have not been conclusive [ 141 ]. In addition, assessment of potential neuropathology in this region could further define the meaning of the increased volume. Males likewise showed changes in fractional anisotropy values for a range of brain structures, most of which involved reductions, including nuclei of the amygdala, the hypoglossal nucleus, the pretectal region, and the trigeminal nerve, all of which are ultimately directly or indirectly interconnected via the trigeminal nerve. The reductions in FA could suggest the potential for demyelination, inflammation, edema and axonal loss. Moreover, these findings are consistent with a significant reduction in volume of the trigeminal nerve that was also observed in males. Other studies of Fe inhalation suggest potential sources of the deficits seen here. For example, Fe inhalation was found to increase numbers of activated microglial cells and levels of Il-1β in olfactory bulb of adult female mice after a 6 hr/day, 5 days/week for 5 week exposure to 200 µg/m 3 iron-soot inhalation that included 40 µg/m 3 of Fe 2 O 3 nanoparticles [ 107 ]. Inhaled Fe nanoparticles have also been reported to produce focal damage to the myelin sheath of a nerve fiber in the olfactory bulb [ 142 ], consistent with the fact that Fe is requisite for myelination [ 143 ]. Interestingly, no effects on brain connectivity were observed in either Fe exposed male or female brains. Future studies are needed to evaluate if a more protracted exposure or prolonged aging would reveal effects of Fe inhalation on the neural connectome. Increased oxidative stress is another feature seen in neurodegenerative diseases including AD [ 144 ] and PD [ 145 ] as caused by impairments in antioxidant capacity [ 146 , 147 ]. Evidence for such alterations was seen in both sexes. In the case of females, this included increases in striatal glutathione even at 48 hr post exposure, but not seen post-behavioral testing. Typically, reductions in glutathione have been associated with mild cognitive impairment and cognitive decline in AD [ 144 , 148 ] that are considered to promote Aβ deposition and tau phosphorylation [ 149 ] and lead to apoptosis [ 150 ]. In the current study, the increase measured in females shortly after the end of the exposure period could represent an adaptive or compensatory response. Correspondingly, in a study of Fe exposure in SH-SY5Y cells, a biphasic GSH response occurred with increases followed by decreases, as Fe exposure concentration increased [ 151 ]. Of further note, however, were the concurrent increases in female striatal levels of glutamate and its precursor, glutamine, both of which are involved in ferroptotic processes, a form of cell death arising from Fe accumulation and consequent oxidative stress and lipid peroxidation. Specifically, glutamate inhibits cystine uptake by the cystine/glutamate antiporter requisite to the production of glutathione [ 152 ]. In the case of males, glutathione increases were also seen, but not until post-behavioral testing. In conjunction with the differences in outcomes of Fe inhalation by sex, the differences in timing of the increases in glutathione suggest the potential for sex differences in antioxidant response/timing that likely contributes to the sex differences in neurodegenerative disease prevalence/phenotypes. Such differences are consistent with known sex differences in redox homeostasis in brain [ 153 ]. Clearly, additional markers of Fe-induced oxidative stress and ferroptosis will be required to determine the extant mechanisms underlying these changes and their inter-relationships. The sex-related differences in response to Fe seen in this study are consistent with a significant literature documenting sex differences in Fe handling and response [ 1 , 92 , 154 , 155 ]. Of particular relevance to the issue of critical periods of exposure, our prior studies have shown that male brain appeared to be more sensitive to inhaled Fe than female brain when such exposures were carried out developmentally in conjunction with exposures to SO 2 , another component of AP, which has been shown to enhance the uptake of Fe into the central nervous system [ 156 ]. Under those conditions, effects of inhalational exposures of C57Bl/6J mice to much lower levels of Fe nanoparticles (1 ug/m 3 ) in conjunction with SO 2 (500 ppb from postnatal days 4–7 and 10–13 (human third trimester brain equivalent; [ 157 ]) for 4 hr/day, were particularly dramatic in males, and included a marked brain metal dyshomeostasis in frontal cortex along with striatal excitatory:inhibitory (glutamate) imbalance and marked increases in levels of dopamine and metabolites, concurrently with reductions in serotonin and metabolites at postnatal day 14 [ 158 ]. Consequently, in addition to sex, timing of exposure and potential cumulative exposures, the chemical speciation of the Fe in air pollution is an additional modifier of neurotoxicity. As previously noted, AP that includes Fe as a contaminant represent a lifelong exposure. Whether early exposures result in developmental reprogramming of brain systems with long term consequences or whether and how the profile of effects observed in the current study would change with more protracted or more cumulative Fe inhalation exposures are all as yet unknown. Findings from the current study underscore the need for defining vulnerable periods of exposure, and assessment of both cumulative exposures and of the longitudinal trajectory of Fe-related brain impacts, as well as the mechanisms that contribute to sex and brain region differences in vulnerability to inhaled Fe. CONCLUSIONS The current study demonstrates that inhaled exogenous Fe UFPs and reach brain, where, in female brain it can produce features characteristic of AD, while in male brain it can alter volume of regions involved in PD. Levels of UFPs, considered the most reactive component of AP, are not regulated by the United States Environmental Protection Agency given monitoring complications and based, in part, on the assumption that UFP concentrations would decline with regulated reductions in levels of PM 2.5 . However, this assumption has not necessarily proven to be the case, which in fact, can be inversely related [ 159 , 160 , 161 , 162 , 163 ]. Findings from the current study underscore the need to further understand the potential neurotoxic consequences of metal constituents within UFPs, especially Fe as it is an essential, redox active metal that has already known to be elevated in brain in numerous neurodegenerative diseases. Further studies of inhaled Fe could yield information ultimately critical to understanding mechanisms of neurodegeneration. Additionally, evidence from the current studies suggests that regulation of Fe levels in AP, and, in particular, in areas of high concentrations such as subways, might provide public health protection against a broad set of neurodegenerative diseases and disorders. Declarations Ethics approval and Consent to Participation - This study was carried out in accordance with relevant guidelines and regulations. All mice used were treated according to protocols approved by the University of Rochester Medical Center Institutional Animal Care and Use Committee and Committee on Animal Resources (approval #102208/2010-046E) and in accordance with NIH guidelines. Consent for publication - Not applicable Competing Interests - The authors declare that they have no competing interests. Funding - Supported by NIH grants R01 ES032260 (Cory-Slechta, PI), R35 ES031689-01A1 (Cory-Slechta, PI) and P30 ES001247 (Lawrence, PI). Author Contribution DAC-S, MS and GO designed the study and prepared the manuscript; DC and RG carried out and monitored all exposures; JVG, AM, KW KC and EM carried out measurements of outcome variables; UG carried out Fe speciation analyses in TEM grids and olfactory bulb; J.A.G. and K.X carried out the magnetic resonance histology and diffusivity analyses. All authors read and approved the final manuscript. Acknowledgement We would like to thank the University of Rochester Electron Microscopy Shared Resource Laboratory for their assistance in processing tissue for Electron Microscopy and Spectroscopy techniques. Availability of Data and Materials - The datasets used and/or analyzed in the current study are available from the corresponding authors upon reasonable request. References Baringer SL, Simpson IA, Connor JR. 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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-5314480","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370416655,"identity":"7504b0ec-27ea-4811-920d-ab624ff51550","order_by":0,"name":"Jithin V. George","email":"","orcid":"","institution":"University of Rochester Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Jithin","middleName":"V.","lastName":"George","suffix":""},{"id":370416656,"identity":"4ca32a5b-1dd2-4e2f-980a-0484ef2f96e7","order_by":1,"name":"Kathryn J. 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Cory-Slechta","email":"","orcid":"","institution":"University of Rochester Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Deborah","middleName":"A.","lastName":"Cory-Slechta","suffix":""},{"id":370416667,"identity":"1866f438-90cd-4345-ade9-ce653663f0e2","order_by":12,"name":"Marissa Sobolewski","email":"data:image/png;base64,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","orcid":"","institution":"University of Rochester Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Marissa","middleName":"","lastName":"Sobolewski","suffix":""}],"badges":[],"createdAt":"2024-10-22 22:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5314480/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5314480/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12989-025-00622-z","type":"published","date":"2025-05-01T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68290764,"identity":"cd8e7602-e7ad-469c-8be9-ac3679fcb115","added_by":"auto","created_at":"2024-11-05 17:12:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38148,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of experimental design. 32 young adult male and 32 young adult female mice were exposed via inhalation to 100 ug/m\u003csup\u003e3\u003c/sup\u003e Fe oxide or filtered air for 2 hr/day for 5 days/week (M-F) for a total of 20 exposure sessions. Brains were collected from a subset of mice from each sex/treatment group at 2 days post exposure for Fe speciation analyses, magnetic resonance histological analyses and quantification of brain neurotransmitter levels. Six mice from each sex/treatment group were subjected to behavioral testing over approximately 6 months after which brains were collected for determination of neurotransmitter levels and quantification of Ab42 and Tau.\u003c/p\u003e","description":"","filename":"Figure1.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/4301a5be3506742e578c61d6.png"},{"id":68292083,"identity":"1c800f04-a3f8-4b2d-984d-a7370968cdf1","added_by":"auto","created_at":"2024-11-05 17:28:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eMean ± standard deviation mass concentrations (ug/m\u003csup\u003e3\u003c/sup\u003e) and particle counts (#/cm\u003csup\u003e3\u003c/sup\u003e) of Fe oxide across the 20 exposure sessions. \u003cstrong\u003eB.\u003c/strong\u003e Mean particle diameter (nm) across the 20 exposure sessions.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/1c5c8c28f3b8f43e86bce346.png"},{"id":68291007,"identity":"854dad6e-a879-4b09-9421-1831493a69c8","added_by":"auto","created_at":"2024-11-05 17:20:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2027232,"visible":true,"origin":"","legend":"\u003cp\u003eFe speciation is illustrated using STEM imaging.\u0026nbsp; Top “A”:\u0026nbsp; TEM Grid Fe Speciation. (a) STEM image shows Fe3O4 nanoparticle agglomerate with corresponding elemental maps for Fe and O in (b) and (c).\u0026nbsp; High resolution STEM in (d) and (e) shows polycrystalline Fe3O4 domains and a thin rim consisting of hematite (Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) approximately 1-2 nm wide with low density (red arrows). Electron diffraction patterns are illustrated for magnetite core (f) and hematite rim (g).\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/a1358a61b51e38326a8918da.png"},{"id":68291006,"identity":"9916dac7-a0ee-4d45-aa34-fba3f8d04744","added_by":"auto","created_at":"2024-11-05 17:20:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2214687,"visible":true,"origin":"","legend":"\u003cp\u003eGroup mean ± standard error levels of glutamatergic, serotonergic and dopaminergic\u0026nbsp; neurotransmitters (area/weight) (g\u003csup\u003e-1\u003c/sup\u003e) in striatum of female (top row) and male mice (bottom row) exposed to filtered air (Air; gray shaded area) or Fe nanoparticles (Fe; symbols) measured two days post termination of exposure. GABA = gamma aminobutyric acid; Gln = glutamine; Glu = glutamate; Tyr = tyrosine; DA = dopamine; DOPAC = 3,4-Dihydroxyphenylacetic acid; HVA = homovanillic acid; NE = norepinephrine; Kyn = kynurenine; Trp = tryptophan; 5HTP = 5-hydroxytryptophan; 5HT = serotonin; 5HIAA = 5-hydroxyindoleacetic acid.\u0026nbsp; * indicates statistically significant at p ≤0.05; ~ indicates p value ≤0.10.\u003c/p\u003e","description":"","filename":"Figure4.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/22df7d33551861dbe2fef83f.png"},{"id":68290761,"identity":"8e382686-6eaf-4266-86cc-6939f5b7aae9","added_by":"auto","created_at":"2024-11-05 17:12:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1973095,"visible":true,"origin":"","legend":"\u003cp\u003eGroup mean ± standard error levels of glutamatergic, serotonergic and dopaminergic\u0026nbsp; neurotransmitters (area/weight) (g\u003csup\u003e-1\u003c/sup\u003e) in cerebellum of female (top row) and male mice (bottom row) exposed to filtered air (Air; gray shaded area) or Fe nanoparticles (Fe; symbols) measured two days post termination of exposure. (area/weight) (g\u003csup\u003e-1\u003c/sup\u003e) in cerebellum of female mice (top row) and male mice (bottom row) exposed to filtered air (Air; gray shaded area) or Fe nanoparticles (Fe; symbols) measured two days post termination of exposure. GABA = gamma aminobutyric acid; Gln = glutamine; Glu = glutamate; Tyr = tyrosine; DA = dopamine; DOPAC = 3,4-Dihydroxyphenylacetic acid; HVA = homovanillic acid; NE = norepinephrine; Kyn = kynurenine; Trp = tryptophan; 5HTP = 5-hydroxytryptophan; 5HT = serotonin; 5HIAA = 5-hydroxyindoleacetic acid. * indicates statistically significant at p ≤0.05; ~ indicates p value ≤0.10.\u003c/p\u003e","description":"","filename":"Figure5.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/a1dbb1b02be125aa1140eec7.png"},{"id":68290772,"identity":"4fb76ef9-3fbd-4ddb-8af7-6f1c556d545c","added_by":"auto","created_at":"2024-11-05 17:12:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":86753,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual specimen diffusivity values (10\u003csup\u003e-3\u003c/sup\u003e x mm\u003csup\u003e2\u003c/sup\u003e/s) in olfactory bulb of male mice exposed to filtered air (blue circles) or Fe nanoparticles (blue asterisks) and female mice exposed to filtered air (red circles) or Fe nanoparticles (red asterisks) as assessed in dMRI from specimen perfused two days post termination of exposure. * Indicates statistically significant at uncorrected p ≤0.05 for each diffusivity value grouping. ** Indicates statistically significant at uncorrected p ≤0.01 for each diffusivity value grouping. Black is significance determined by the 2-Way ANOVA with interactions analysis. Red (female) and Blue (male) indicates significance as determined by the stratified Kruskal Wallis non-parametric ANOVAs.\u003c/p\u003e","description":"","filename":"Figure6.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/658b8a872e065bf1654b8d9d.png"},{"id":68290773,"identity":"797f52e5-d72c-494a-ba9b-bc1d8344f585","added_by":"auto","created_at":"2024-11-05 17:12:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1987613,"visible":true,"origin":"","legend":"\u003cp\u003eGroup mean ± standard error levels of phosphorylated tau (left column), total tau (middle column) and Ab42 (right column) (pg/mL) in frontal cortex (top row) and hippocampus (bottom row) of male (blue) and female (red) mice exposed to filtered air (Air) or Fe nanoparticles (Fe). * indicates statistically significant at p ≤0.05.\u003c/p\u003e","description":"","filename":"Figure7.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/bd55e323773c21becced38de.png"},{"id":68291008,"identity":"4dca93ae-7079-4b52-82bb-6152ab2b323a","added_by":"auto","created_at":"2024-11-05 17:20:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1740879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLeft Column: \u003c/strong\u003eGroup mean ± standard error levels of time spent with object/total time spent exploring in session one (left column) of the NOR paradigm of left and right objects in females (top left) and males (bottom left) exposed to filtered air (Air) or to Fe nanoparticles (Fe). \u003cstrong\u003eRight Column:\u003c/strong\u003e novel object recognition index in NOR session 2 for males (blue) and females (red) exposed to filtered air (Air) or to Fe nanoparticles (Fe). * indicates statistically significantly different from Air at p ≤0.05.\u003c/p\u003e","description":"","filename":"Figure8.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/ec4bc4822f31a80c4e07aa4a.png"},{"id":68290765,"identity":"3a128128-8396-483c-a386-a3f2a929bd5c","added_by":"auto","created_at":"2024-11-05 17:12:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":569181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTop row: \u003c/strong\u003eGroup mean ± standard error of percent errors on the RAM in males (left) and females (right) exposed to filtered air (closed circles) or to Fe nanoparticles (open circles) across days of measurement. * indicates statistically significantly different from Air at p ≤0.05.\u003cstrong\u003e Bottom row:\u003c/strong\u003e Correlation of hippocampal phosphorylated tau levels (pg/mL) with percent errors during session 3 of RAM in female mice.\u003c/p\u003e","description":"","filename":"Figure9.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/bd5d1bd2b9c9732e7d200dce.png"},{"id":68291009,"identity":"a951b243-b156-48ab-a4a7-45fd80c46784","added_by":"auto","created_at":"2024-11-05 17:20:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":369298,"visible":true,"origin":"","legend":"\u003cp\u003eGroup mean ± standard error levels of glutamatergic, serotonergic and dopaminergic\u0026nbsp; neurotransmitters (area/weight) (g\u003csup\u003e-1\u003c/sup\u003e) in striatum of female (top row) and male mice (bottom row) exposed to filtered air (Air; gray shaded area) or Fe nanoparticles (Fe; symbols) measured after behavioral testing. GABA = gamma aminobutyric acid; Gln = glutamine; Glu = glutamate; Tyr = tyrosine; DA = dopamine; DOPAC = 3,4-Dihydroxyphenylacetic acid; HVA = homovanillic acid; NE = norepinephrine; Kyn = kynurenine; Trp = tryptophan; 5HTP = 5-hydroxytryptophan; 5HT = serotonin; 5HIAA = 5-hydroxyindoleacetic acid.\u0026nbsp; * indicates statistically significant at p ≤0.05; ~ indicates p value ≤0.10.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/4b2c75306f62bc4cfa2808e4.png"},{"id":68290767,"identity":"4adea26e-4cf1-4e7f-947e-6ba67f9a0855","added_by":"auto","created_at":"2024-11-05 17:12:03","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1927864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003eGroup mean ± standard error levels of glutamatergic, serotonergic and dopaminergic\u0026nbsp; neurotransmitters (area/weight) (g\u003csup\u003e-1\u003c/sup\u003e) in cerebellum of female (top row) and male mice (bottom row) exposed to filtered air (Air; gray shaded area) or Fe nanoparticles (Fe; symbols) measured after behavioral testing. (area/weight) (g\u003csup\u003e-1\u003c/sup\u003e) in cerebellum of female mice (top row) and male mice (bottom row) exposed to filtered air (Air; gray shaded area) or Fe nanoparticles (Fe; symbols) measured two days post termination of exposure. GABA = gamma aminobutyric acid; Gln = glutamine; Glu = glutamate; Tyr = tyrosine; DA = dopamine; DOPAC = 3,4-Dihydroxyphenylacetic acid; HVA = homovanillic acid; NE = norepinephrine; Kyn = kynurenine; Trp = tryptophan; 5HTP = 5-hydroxytryptophan; 5HT = serotonin; 5HIAA = 5-hydroxyindoleacetic acid. * indicates statistically significant at p ≤0.05; ~ indicates p value ≤0.10.\u003c/p\u003e","description":"","filename":"Figure11.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/d776e89f76cfaade971a91c4.png"},{"id":68291010,"identity":"e9b5f334-02c5-4bdd-81ee-8f828ed3798f","added_by":"auto","created_at":"2024-11-05 17:20:03","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1979470,"visible":true,"origin":"","legend":"\u003cp\u003eGroup mean ± standard error levels of striatal (top row) and cerebellar (bottom row) transulfuration markers of females (left column) and males (right column) exposed to filtered air (gray shaded area) or to Fe measure at 2 days post exposure (pre) and following behavioral testing (post) including methionine, h-cysteine, cysteine and glutathione (GSH). * indicates statistically significant at p ≤0.05; ~ indicates p value ≤0.10.\u003c/p\u003e","description":"","filename":"Figure12.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/a83427e88fe4ab4e7b78662d.png"},{"id":81987691,"identity":"ed6d4db1-4e5a-417e-8fa2-494633f507e6","added_by":"auto","created_at":"2025-05-05 16:04:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16467105,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/7b37af76-49cd-42c9-a71d-e7eeb7b96196.pdf"},{"id":68290769,"identity":"2cfce1f5-d729-46b4-8d0e-991dbfcc2d06","added_by":"auto","created_at":"2024-11-05 17:12:03","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":411456,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5314480/v1/dd08a78d297bd77e9584ca8f.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Brain Iron Accumulation in Neurodegenerative Disorders: Does Air Pollution Play a Role?","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eExcess brain iron (Fe) accumulation is seen in numerous neurodegenerative diseases and disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and, given the redox activity of Fe, therefore often considered to play a key role in the etiology of these disorders [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In Alzheimer\u0026rsquo;s disease (AD), for example, a female-biased disorder characterized by the accumulation in brain of beta amyloids and tau and with dementia and cognitive decline, brain Fe levels are elevated above those associated with normal aging, even when concurrent elevations in serum Fe are not seen [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cortical Fe levels have been found to correlate with the Braak staging of the severity of AD [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similarly, Fe accumulation is found in substantia nigra and the red nucleus [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] even in early stages of Parkinson\u0026rsquo;s Disease (PD), a male-biased neurodegenerative disease resulting from the loss of dopamine neurons in substantia nigra that lead to motor impairments [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Further, elevated brain Fe has been shown to be negatively associated with general cognitive ability, and positively associated with cognitive aging in normal elderly individuals and even in children [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and cognitive impairments are found in both AD and PD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the historical recognition of elevated brain Fe content in neurodegenerative diseases and disorders, the basis for this accumulation is not known, including in what forms excess Fe is incorporated at cellular levels. Excess brain Fe accumulation has typically been ascribed to e.g., dysregulation of Fe homeostasis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] or breakdown of the blood brain barrier [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. What has never been considered, however, is the hypothesis that exposure to Fe via air pollution (AP) may play a primary role, elevating exogenous Fe levels in brain, and promoting dysregulation of endogenous Fe homeostasis in response to uptake of exogeneous Fe-laden nanoparticles. Indeed, Fe constitutes one of the most abundant metal contaminants of AP [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and exposures to AP are life-long. AP is a dynamic and complex mixture of gases (e.g., SO\u003csub\u003e2\u003c/sub\u003e and NO\u003csub\u003ex\u003c/sub\u003e) and particulate matter (PM), with PM defined by particle size (i.e., coarse particles or PM\u003csub\u003e10\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026le;10 um, fine particles or PM\u003csub\u003e2.5\u003c/sub\u003e=\u0026le;2.5 um; ultrafine particles or UFP or PM\u003csub\u003e0.1\u003c/sub\u003e=\u0026le;100 nanometers) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The UFP component is considered the most reactive component of AP given its greater surface area/mass ratio for adherence of contaminant particles that include metals such as Fe and other trace elements [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Of particular note UFPs, including metal oxides like Fe, are able to travel directly from deposits in the nose into the brain via olfactory axons and thereby bypass the blood brain barrier [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], which could explain the corresponding lack of any consistent elevations in serum Fe in AD and PD [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Further, mechanisms to remove excess Fe from brain are not apparent [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and were likely not needed prior to industrialization.\u003c/p\u003e \u003cp\u003eIn accordance, AP exposures have been associated in epidemiological studies and in systematic reviews with multiple neurodegenerative diseases, including both AD and PD [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. More specifically, exposures to the PM\u003csub\u003e2.5\u003c/sub\u003e (size fractions \u0026le;2.5 um), component of AP as available through monitoring, have been associated with reduced memory and processing speed [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and increased cognitive impairment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], with some studies suggesting a more pronounced impact on cognitive decline in women [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Both PD and AD diagnoses has been correlated with levels of NO\u003csub\u003ex\u003c/sub\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], a marker of traffic density, and with levels of PM\u003csub\u003e2.5\u003c/sub\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It is important to note that UFP is a component of PM\u003csub\u003e2.5\u003c/sub\u003e. AP exposures have also been reported to alter brain structure, with reports of reductions in both white matter and gray matter volumes (reviewed in [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]) and in hippocampal volume [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and with smaller deep-gray matter brain volumes and white matter volumes in frontal and temporal lobes, reductions in size of the corpus callosum, ventriculomegaly, and increases in brain infarcts [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnimal studies support these epidemiological associations of AP with neurodegenerative disease [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and provide mechanistic links. A 15-week exposure to re-suspended urban PM\u003csub\u003e2.5\u003c/sub\u003e nanoparticles of wild type or EFAD mice that carry either ε3 or ε4 transgenes for human APOE, a gene known to enhance vulnerability to AD, in conjunction with five familial AD mutations, resulted in increased Aβ levels [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, both wild type and transgenic mice exhibited selective hippocampal neuronal atrophy. Exposures of wild type and TgF344-AD rats to real-world traffic-related AP resulted in AD neuropathology, including increased neuroinflammation and amyloid plaque deposition, higher levels of hyperphosphorylated tau, neuronal cell loss, and cognitive deficits in an age-, genotype-, and sex-dependent manner [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Real-world, concentrated UFP exposure resulted in alterations in memory domains [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], with altered microglial morphology and elevated phosphorylated tau in 3xTgAD mice [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In addition, exposures of aged rats for 3 months to PM\u003csub\u003e2.5\u003c/sub\u003e produced motor impairments and reduced tyrosine hydroxylase expression in olfactory and nigrostriatal circuits, increased radial diffusivity in MRI assessments and enhanced age-related demyelination [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In another study, an 8-week exposure to ambient fine or ultrafine particles reduced locomotion in rats in conjunction with decreased dopamine uptake [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOf the many metals/trace elements in AP, Fe is often one of the most abundant [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Fe levels in air measured at 13 sites in the U.S. averaged 108 ng/m\u003csup\u003e3\u003c/sup\u003e with a range of 41\u0026ndash;240 ng/m\u003csup\u003e3\u003c/sup\u003e [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Furthermore, highly racially segregated counties sustain higher mass concentrations of fine particulate metals, indicating the importance of evaluating specific metal constituents of AP [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. While collectively summed U.S.-wide ambient maximums indicate levels of 1210 \u0026micro;g/m\u003csup\u003e2\u003c/sup\u003e/day of Fe that include broad spatial variation based on emission sources [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e63\u003c/span\u003e], Fe levels can be concentrated in particular areas and reach extraordinarily high concentrations. For example, ambient PM exposures in underground U.S. subway systems were recently estimated to range from 112\u0026thinsp;\u0026plusmn;\u0026thinsp;46.7 to 779\u0026thinsp;\u0026plusmn;\u0026thinsp;249 \u0026micro;g/m\u003csup\u003e3\u003c/sup\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and in other studies from 141\u0026thinsp;\u0026plusmn;\u0026thinsp;81.1 to 329\u0026thinsp;\u0026plusmn;\u0026thinsp;116 \u0026micro;g/m\u003csup\u003e3\u003c/sup\u003e [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. While Fe in ambient AP from different sources can occur in water-soluble (Fe2\u003csup\u003e+\u003c/sup\u003e) forms [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e67\u003c/span\u003e], it can also be present as Fe (II, III) oxide: i.e., magnetite (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) and hematite (Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Analysis of Fe speciation is critical, as solubility and bioavailability of these metals influences redox potential that can produce oxidative stress. Magnetite nanoparticles, a mixed oxide with Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e, have been found in human postmortem brain samples [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e75\u003c/span\u003e], and brain magnetite has been linked to the incidence of AD [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e77\u003c/span\u003e] and found to be directly associated with both Aβ plaques and tau tangles [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThat Fe-contaminated AP may be a source of excess brain Fe is suggested by two studies. In one, an abundant presence of magnetite (Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e iron oxide) nanoparticles approximately 10\u0026ndash;150 nm in size, interpreted as being consistent with an exogenous rather than endogenous source of Fe formation based on crystal morphologies that pinpoint to high temperature formational mechanisms such as during coal combustion, was identified in frontal cortex of brains from AD patients [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Additionally, a recent report found an accumulation of ambient black carbon particles (a component of air pollution particulate matter) in thalamus, prefrontal cortex and olfactory bulb and hippocampus in post-mortem brains from individuals with neuropathologically confirmed Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, these data highlight the need to evaluate the neurotoxicity of inhaled Fe-oxide nanoparticles in AP and their potential relationship to neurodegenerative disorders such as AD and PD. For that purpose, the current study sought to confirm in a mouse model that: (i) brain uptake of exogenous Fe nanoparticles via inhalation exposure occurs, and (ii) to examine the hypothesis that inhalation of such Fe oxide nanoparticles could reproduce features of neurodegenerative diseases/disorders in a mouse model. The focus of the consequences of the exposures included some features unique to AD and PD, as well as some features shared across neurodegenerative diseases and disorders [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Sex-related differences in brain Fe metabolism as well as in accumulation with aging have been reported and may contribute to sex-bias in neurodegenerative disorders [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e85\u003c/span\u003e], and thus sex-related differences in outcome were also hypothesized.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAnimals and Husbandry\u003c/span\u003e: Male and female young adult (10 weeks old) C57Bl/6J mice from Jackson labs were placed in same-sex pairs and housed in standard mouse caging maintained at 22+-2 \u0026deg;C on a 12-h light-dark cycle (lights on at 06:00) at the University of Rochester Medical Center. Cages were provided with approximately 3 mm high performance bedding (BioFresh), \u003cem\u003ead libitum\u003c/em\u003e standard rodent chow (LabDiet Autoclavable Diet 5010) and water. Male and female mice were randomly assigned to either control or Fe-exposure groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, a subset of mice were euthanized at 48 hours post final Fe exposure and brains collected for Fe speciation analyses and magnetic resonance histological (MRH) analyses (n\u0026thinsp;=\u0026thinsp;3/4 sex/treatment group) and for analyses of neurotransmitter levels (n\u0026thinsp;=\u0026thinsp;6/sex/treatment group) in frontal cortex and cerebellum. An additional subset of mice were randomly selected for behavioral assessments and brains collected following euthanization at the termination of behavioral testing at approximately postnatal day 270, i.e., approximately 6 mos post-exposure, for evidence of protein aggregation (n\u0026thinsp;=\u0026thinsp;12/sex/treatment group) and brain neurotransmitter levels. All mice used in the experiment were weighed every other day to monitor for signs of exposure-related systemic toxicity and treated humanely to alleviate suffering where possible in accordance with protocols approved by the Institutional Animal Care and Use Committees at the University of Rochester. For all mouse behavioral experiments and biochemical assessments, mouse/samples were counterbalanced by treatment group and sex to ensure any temporal or experimental variation was distributed across all groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eFe Oxide: Electric Spark Generation and Exposure\u003c/span\u003e: Mice were exposed to Fe oxide via inhalation in compartmentalized whole body exposure chambers. For this study, the intended Fe concentration was 100 \u0026micro;g/m\u003csup\u003e3\u003c/sup\u003e which was chosen to be below the range of values cited for subway Fe levels. Exposures were carried out for 2 hours per day [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Mice were exposed for 5 days/week (M-F) over one month (July 8th, 2021 to August 6th, 2021) for a total of 20 exposure sessions. This exposure paradigm was roughly designed based on Organisation for Economic Co-Operation and Development (OECD) subacute inhalation toxicity study guidelines [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e87\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhole-body inhalation exposures were conducted in the University of Rochester Inhalation Core Facility in single-house 30L stainless-steel reinforced Lexan exposure chambers. Control mice were exposed to HEPA-filtered air and experimental mice were exposed to Fe-oxide UFP particles generated by electric spark discharge in argon between two 99.99% pure iron rods (3N5 Purity, ESPI Metals, Ashland, OR, USA) using a GFG-1000 Palas generator (Palas GmbH, Karlshrue, Germany) in an argon atmosphere. Airborne particles were passed through a deionizer so that particles reached Boltzmann equilibrium charge. Particle number concentration was controlled by spark discharge frequency. Aerosol number concentration and particle size were monitored in real-time via a Condensation Particle Counter (CPC, model 3022, TSI Inc, St Paul, MN, USA) and Scanning Mobility Analyzer (SMPS, model 3934 TSI Inc, St Paul, MN, USA) respectively.\u003c/p\u003e \u003cp\u003eThe Fe-oxide particles (FeO, Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles) were generated by adding a low flow of oxygen (~\u0026thinsp;50 mL/min) into the argon flow (~\u0026thinsp;5 L/min) which then entered the spark discharge chamber. An O\u003csub\u003e2\u003c/sub\u003e sensor (MAXO2 -250E, Maxtec, Salt Lake City, UT, USA) confirmed the maintenance of an oxygen concentration of 21% in the exposure chamber. This procedure produced particle sizes exclusively in the ultrafine size range with a count median diameter (CMD) of approximately 30\u0026ndash;34 nm. Mass concentrations were determined gravimetrically by filter weight (25mm, Emfab Membrane Filters, Pall Life Sciences, Port Washington, NY) collected twice daily (5 L/min for 60 min., 300L total volume) from the filtered air and ultrafine Fe-oxide particle exposure chambers and secondarily determined using ICP-MS data. Electrostatic precipitation was used to collect particles on transmission electron microscopy (TEM) grids made of copper (add grid details).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eMagnetic Resonance Histology\u003c/span\u003e: Magnetic resonance histology (MRH) was performed using methods previously described fully [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e88\u003c/span\u003e], conducted similarly to magnetic resonance imaging only these evaluations are conducted post-mortem. Briefly, mice were perfusion-fixed using an active stain of buffered formalin and Prohance, a Gd contrast agent used to reduce the spin lattice relaxation time (T1) with imaging using a 9.4T vertical bore magnet [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. These specimens use the same diffusion weighted imaging protocol that has been ported to a 7T horizontal bore magnet with similar, gradient, and rf coils. A 3D spin echo Steskal Tanner sequence was used with TR/TE\u0026thinsp;=\u0026thinsp;100/15.8 ms with isotropic spatial resolution of 35 microns. Forty-six 3D volumes were acquired with b values of 3000 s/mm2 with b vectors that uniformly sample the unit sphere. Five baseline (b0) volumes were acquired. All data was acquired with compressed sampling using an acceleration of 8X and reconstructed using the iterative methods described in [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Labels were applied using the methods described in [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. The labels (r1CCFv3) are consistent with the Allen Brain Atlas common coordinate framework with modifications to accommodate quantitative connectomics. The b0 volumes were registered together. The 46 diffusion weighted volumes were registered to this average baseline to reduce the consequences of eddy current. The resulting 4 dimensional volume was post processed in DSI Studio (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dsi-studio.labsolver.org/\u003c/span\u003e\u003cspan address=\"https://dsi-studio.labsolver.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) yielding the following quantitative scalar images: axial diffusivity (AD), mean diffusivity (MD), radial diffusivity (RD), fractional anisotropy (FA), color fractional anisotropy (clrFA) all of which provide insight into the tissue cytoarchitecture [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e90\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral Assessment:\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLocomotor Activity\u003c/span\u003e \u003cb\u003e\u0026ndash;\u003c/b\u003e Spontaneous locomotor activity was measured in one 60-minute session in chambers (27.3 cm x 27.3 cm x 20.3 cm) that contained 48-channel infrared photobeams (Med Associates Inc., St. Albans, Vermont). Photobeam breaks were recorded across five-minute intervals for 60 minutes using measures of stereotypic, vertical, and ambulatory movements as well as ambulatory distance and time in center vs. edge zones. Stereotypic counts were defined as localized movement, i.e., the number of beam breaks within a 2x2 inch photobeam box when non-ambulatory. Vertical counts were defined as the total time that z-axis photobeams or photobeams that were 7 cm above the floor of the locomotor box were broken. Ambulatory counts were defined as the number of photobeam breaks during ambulatory movement, and ambulatory distance was defined as the differences in angular movements. Time in zone was defined as the total time spent within a given zone. Resting time was defined as time spent with no new photobeam breaks. Zone entries were defined as entry of all four paws into a given zone. The locomotor arena was broken into two zones: the center zone (center 15.7 x 15.7 cm square) or edge zone (space between center square and arena boundaries).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eNovel Object Recognition Memory (NOR\u003c/span\u003e \u003cb\u003e) \u0026ndash;\u003c/b\u003e Following the locomotor activity session, NOR assessment was carried out. NOR consisted of two sessions conducted in an open plexiglass arena (30.5 cm x 30.5cm x 30.5cm). During the first session, mice were placed individually into the test chamber containing two small round white knobs secured to the chamber floor, and were allowed to explore the chamber and objects for 10 min. The purpose of this initial session was to allow mice to become familiar with the sample objects and for assessment of potential side preference and overall exploration patterns across exposure groups. The second session occurred 24 h after the first session to assess memory of the sample objects, premised on the ability of mice to detect and prefer novel stimuli. During the second session, mice were returned to the testing chamber, which now contained one small round, white knob (sample object) and one small square, black knob (novel object) in place of the prior white knob. Position of the novel object within the chamber (right or left side) was counterbalanced across treatments and subjects to preclude side bias. Both sessions were videotaped and scored using Noldus software (The Observer XT, Noldus) by a trained observer blinded to treatment condition. Object exploration was defined as a mouse being oriented toward the object with its head crossing a pre-marked 2 cm circle surrounding the object. Object recognition was analyzed using three different indices which control for differences in overall exploration across mice: duration index (total novel exploration time / [total novel time\u0026thinsp;+\u0026thinsp;total sample time]), bout index (total novel bouts / [total novel bouts\u0026thinsp;+\u0026thinsp;total sample bouts]), and time-per-bout index (average novel time per bout / [average novel time per bout\u0026thinsp;+\u0026thinsp;average sample time per bout]).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eRadial Arm Maze (RAM)\u003c/span\u003e \u003cb\u003e\u0026ndash;\u003c/b\u003e Following NOR testing, RAM performance was evaluated. The radial arm maze consisted of 8 arms emanating from a center arena. Mice were gradually food restricted until they reached 85% of their free-feeding body weight. Mice were then habituated to the radial arm maze in two sessions separated by 24 hours. In the first habituation session, two mice from the same cage were placed in the maze and allowed to freely explore. For the second habituation session, mealworms were placed at the end of each arm of the maze and then mice were individually introduced to the maze to freely explore for five minutes. Experimental sessions began 48 hours after the second habituation session.\u003c/p\u003e \u003cp\u003eIn subsequent experimental sessions, mealworms were placed in odd- or even-numbered arms, with placement counterbalanced by sex and treatment group. Mice were then placed in the center of the maze for five seconds with all arm doors closed. Then, doors were simultaneously raised and mice allowed to freely explore the maze. Arm entry was defined as all four paws of the mouse within an arm, past the arm door. If a mouse entered an arm, the door to the arm closed until the reward was completely consumed or for a total of five seconds. This process was repeated until all rewards were consumed or until the ten-minute maximum session time was reached, whichever occurred first. The maze was thoroughly cleaned with disinfectant between each test session. Male mice underwent 5 test sessions, while female mice underwent 3 test sessions due to disruptions by construction-related activity. Number of correct entries, number of incorrect entries or working memory errors (defined as repeat entries to arms after reward was already consumed), and time to obtain all rewards were recorded. Percent error was calculated as the ratio of number of incorrect entries to total number of entries multiplied by 100.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAβ42, Total and pS199 Tau Protein Quantification:\u003c/h3\u003e\n\u003cp\u003eAt sacrifice (approximately postnatal day 270), hippocampus, frontal cortex, and striatum were dissected out and processed for ELISA analysis. Amyloid beta 42, total tau and pS199 tau concentrations were measured in duplicate using commercially available immunoassay kits (Invitrogen, Waltham, MA, USA, catalogue\u0026ensp;KMB3441, KMB7011 and KMB7041 respectively) according to the manufacturer\u0026rsquo;s specifications. Samples were read on a SynergyH1 Hybrid Reader (BioTek, Winooski, VT, USA) with Gn5 2.01 software. Sample replicates with coefficient of variation (COV) higher than 15% were excluded from the analysis. Standard curve CVs fell below 10%.\u003c/p\u003e\n\u003ch3\u003eBrain Neurotransmitter Levels\u003c/h3\u003e\n\u003cp\u003eStriatal and cerebellar concentrations of dopamine (DA), 3-4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), tyrosine (Tyr), norepinephrine (NE), glutamine (Gln), glutamate (Glu), gamma-aminobutyric acid (GABA), tryptophan (Trp), kynurenine (Kyn), serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) were measured in brains collected 48 hr post exposure to Fe and in another subset of mice at approximately PND270. For this purpose, hemisected brain tissue was thawed and diluted with 75 \u0026micro;L of ice-cold acetonitrile (50%, v/v) and homogenized via sonication for 10 seconds (SLPe digital sonifier, Branson Ultrasonics Corp., Danbury, CT.). Samples sat for ten minutes after which homogenates were collected and centrifuged at 10,000g for 20 mins and 4\u0026deg;C. The new supernatant was then collected and stored at -80\u0026deg;C until LC-MS analysis.\u003c/p\u003e \u003cp\u003eStock solutions of the above analytes were made at 5 mg/mL in ddH\u003csub\u003e2\u003c/sub\u003eO, except Tyr which was made in 0.2 M HCl. To study endogenous neurotransmitter variations within specific regions, standard solutions were made with ddH2O with analyte concentrations ranging as per prior range-finding studies. The solution was then derivatized using 13C6 benzoyl chloride (BzCl, Sigma Aldrich) as described by Wong et al., to create individual neurotransmitter internal standards. Internal standards were aliquoted and stored at -80\u0026deg;C until LC-MS analysis [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e91\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRight before analysis, internal standard mixtures were thawed, diluted in 50% acetonitrile and mixed with 1% sulfuric acid. This mixture was then added to derivatized samples. Samples were then centrifuged at 16,000g for five minutes and 20 \u0026micro;L of supernatant collected into a LoBind tube (Eppendorf). 10 \u0026micro;L of 100 mM sodium carbonate, 10 \u0026micro;L of 2% BzCl in acetonitrile, and 10 \u0026micro;L of internal standard were added sequentially to the LoBind tube. 50 \u0026micro;L of ddH2O was then added to reduce organic concentration and then, samples were centrifuged to remove any remaining protein pellets. The resulting supernatant was added to an autosampler vial.\u003c/p\u003e \u003cp\u003eLC-MS/MS analysis was carried out by a Dionex Ultimate 3000 UHPLC coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher). Analytes were separated on a Waters Acquity HSS T3 column. The mobile phases were: 10 mM ammonium formate in 0.1% formic acid, and also, acetonitrile. The flow rate was set to 400 \u0026micro;L/min and the column oven was set at 27\u0026deg;C. After 5 \u0026micro;L of each sample was injected, the analytes were separated using a 12-minute multi-step gradient. The Q Exactive Plus was operated in positive mode, and a parallel reaction monitoring method (PRM) was used to detect derivatized molecules. Fragment ions were extracted with a 10 ppm mass error using the LC Quan node of the Xcalibur software (Thermo Fisher). Endogenous analyte peak areas were compared to those of each internal standard to determine relative abundance. These values were then divided by wet weight of the sample and then divided by air control to yield percent of control values. Turnover of neurotransmitters was also calculated including Gln/Glu, Glu/GABA, 5HIAA/5HT, HVA/DA and DOPAC/DA.\u003c/p\u003e\n\u003ch3\u003eFe Nanoparticle Speciation in Brain: Distinguishing Exogenous and Endogenous Fe\u003c/h3\u003e\n\u003cp\u003eFe nanoparticle speciation was identified in olfactory bulb (OB) thin sections using high-resolution scanning/transmission electron microscopy (S/TEM) coupled with spectroscopic elemental mapping. A JEOL 2100 F field emission S/TEM operated at 200 kV with analytic pole piece was used for the OB sections and also to identify the as-synthesized Fe-nanoparticles collected on TEM grids (Ted Pella, Inc. Redding, CA) for comparison. OB thin sections were obtained after brains were extracted and placed in filtered 4% PFA for initial tissue fixation. After dissection (using the right hemisphere), the OB tissues were post-fixed in 2.5% glutaraldehyde using 0.1M sodium phosphate buffer at 4\u0026deg;C followed by fixation in EPON-Araldite epoxy resin and then embedded in epoxy and polymerized at 60\u0026deg;C. All OB tissues were unstained to have a greater contrast of the Fe-nanoparticles with the cellular matrix. Tissue sections were cut to be ~\u0026thinsp;70nm using an ultramicrotome (Boeckeler Instruments, Inc., Tuscon, AZ) and were placed onto nickel formvar/carbon coated slot grids (Ted Pella Inc., Redding, CA) to stabilize the tissue during beam interaction. High-resolution images of Fe nanoparticles in the OBs were recorded with a Gatan Ultrascan 4k CCD camera and data analysis and processing used Gatan Digital Micrograph software (Gatan, Inc.). The S/TEM analysis was coupled with spectroscopic elemental mapping of the Fe nanoparticles in the OB. A GATAN high angle annular dark field (HAADF) detector (Digiscan II) and an Oxfor Aztec EDS system from Oxford Instruments, Oxfordshire, United Kingdom were used. Energy dispersive spectroscopic analysis (EDS) was performed with a GATAN high angle annular dark field detector (HAADF), Digiscan II, Gatan 2000 Image Filter (GIF) with Oxford Aztec EDS software (Oxford Instruments, Oxfordshire, United Kingdom. All S/TEM images were acquired using an analytical probe with 0.17 nm.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eBoth male and female mice were used in all analyses. However, statistical analyses were stratified by sex based on known sex differences in response to Fe and of female bias in AD prevalence [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e93\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e95\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. Brain neurotransmitter levels and levels of Aβ and tau were analyzed using one way analysis of variance (ANOVA). Locomotor activity data was analyzed in five-minute time intervals with repeated measures ANOVA; radial arm maze data was also analyzed by repeated measures ANOVA. NOR data were analyzed via one-way ANOVA separately for each session. Statistical analyses were conducted using JMP Pro 16.0 (SAS Institute Inc., Cary, NC, USA). P values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant, while near significant values (p values\u0026thinsp;\u0026le;\u0026thinsp;0.10) are also indicated. Outliers were first removed if determined by a statistically significant Grubb\u0026rsquo;s test (GraphPad Software Inc., San Diego, CA). Outlier analysis was never iterative, i.e., only one sample removed per treatment group per endpoint if they were confirmed by a Grubb\u0026rsquo;s test.\u003c/p\u003e \u003cp\u003eFor the diffusion MRI metrics and volumes, the statistical tests were again stratified by sex. Kruskal Wallis non-parametric ANOVA was used to determine the significance of change amount of the 180 x 2 regions within the atlas for the axial, radial, and mean diffusion contrasts and volume. For volume, the volume of each region was normalized by the total brain volume of each specimen prior to analysis. Secondly, an omnibus Kruskal Wallis test was performed with groups of Male Air versus Female Air versus Male Fe versus Female Fe to investigate whether the commonly significantly changed regions for the sex stratified changed in the combined model. A posthoc Dunn\u0026rsquo;s test with Sidak correction was used to identify the pairwise comparisons of groups that contributed most to any significant result. P values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant. Eta\u003csup\u003e2\u003c/sup\u003e and Cohen\u0026rsquo;s F were used to quantify strength of the effect size for significant results. The statical calculations of MRI data were done in MATLAB.\u003c/p\u003e \u003cp\u003ePrior to analysis of the MRI metrics and volumes, the values of the left and right structures were combined by summing volumes and performing the weighted mean on diffusion metrics. Parent structures are generated from all potential parents in the RCCF atlas. The volume of each region was normalized by the total brain volume. First, a 2-way ANOVA was carried out with interactions considering sex and iron exposure conditions. This was followed by a Kruskal-Wallis non-parametric ANOVA to consider each sex independently for the iron exposure condition. P values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant. Eta\u003csup\u003e2\u003c/sup\u003e and Cohen\u0026rsquo;s F [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e97\u003c/span\u003e] were used to quantify strength of the effect size for significant results. Using G* Power [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e98\u003c/span\u003e], we estimated the minimal Cohen\u0026rsquo;s F effect size needed to maintain statistical power of 0.8. In the 2-Way ANOVA with interaction testing, the estimated Cohen\u0026rsquo;s F effect size needed to maintain sufficient power is 0.82, while for the stratified by sex Kruskal- Wallis Non-Parametric ANOVA, an effect size of 1.3 is needed to maintain the same power criteria. The statistical calculations of MRI data were done in MATLAB.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFe Exposure Concentrations\u003c/h2\u003e \u003cp\u003eMass concentration averaged 109.5 \u0026plusmn; 24.16 ug/m\u003csup\u003e3\u003c/sup\u003e across the exposures, and mean particle counts averaged 2.01E\u0026thinsp;+\u0026thinsp;06 #/cm\u003csup\u003e3\u003c/sup\u003e across the 20 exposure sessions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The particle diameter remained in the nanoparticle range (43\u0026ndash;52 nm) across the course of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To compare deposition fractions in mice, a multiple-path particle dosimetry model (MPPD; Applied Research Associates, Inc. v3.04) was used, with settings customized to B6C3F1 mouse with subject-specific functional residual capacity (FRC), upper respiratory tract (URT), respiratory rate (RR), and tidal volume (TV). Additionally, density of iron-oxides was set to an average 5.2 g/cm\u003csup\u003e3\u003c/sup\u003e, based on the approximate density of singlet iron oxides (averaged between Fe3O4 and Fe2O3), with a median mass aerodynamic diameter (MMAD) of 0.04812 um as reported above. Using these parameters, estimates of particle deposition were modeled for comparison. The total modeled deposition fraction was 53.84%% with 33.48% depositing in the head, 15.61% depositing in the tracheobronchial region, and 4.7% depositing in the pulmonary region. Of the modeled pulmonary deposition, 16.53% deposited in the central respiratory airway and 3.83% deposited in the peripheral conducting respiratory airways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFe Characterization, Speciation and Translocation to Olfactory Bulb\u003c/h3\u003e\n\u003cp\u003eNasal olfactory uptake of Fe nanoparticles and its speciation was assessed in olfactory bulb, the port of entry into brain and compared to Fe speciation on TEM grids from the inhalation chamber. The STEM analyses with corresponding elemental maps and electron diffraction of the as-synthesized Fe particles are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and provide structural and compositional details for the grid collected Fe speciation from the exposure chamber which formed predominantly magnetite particles, i.e., Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e. Most particles were spherical with polycrystalline domains although some euhedral crystals were recognized (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003ea\u003c/b\u003e). Elemental mapping for Fe and O is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003eb-c\u003c/b\u003e which indicated that oxygen is evenly distributed through the bulk of the particles. At higher resolution, the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e spheres have a narrow band at the surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003ed-e\u003c/b\u003e) that consists of hematite Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, a more oxidized Fe speciation that was further distinguished from the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-rich core region of the Fe particles using electron diffraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003ef-g\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe magnetite particles were also identified in the olfactory bulb after inhalation exposure and examples shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003ea-m\u003c/b\u003e. The olfactory bulb has magnetite present in close proximity to neurons and astrocytes and also next to corpora amylacea which can be distinguished in the OB tissue regions using STEM imaging even in the unstained sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003ea\u003c/b\u003e). Two examples of translocated Fe particles are indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003ea\u003c/b\u003e and the larger Fe agglomerate is magnified in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003eb-e\u003c/b\u003e with corresponding elemental maps for Fe and O. Results indicate that the magnetite (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) particles that translocated to the OB have the same chemical and crystalline structure as the spark-generated particles from the TEM grids. The surface rim of hematite (Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) around the magnetite core that was identified in the TEM grids sample is missing in the olfactory bulb Fe particles which are characterized by a rougher surface layer potentially due to particle-tissue interaction and some bioprocessing of the particles that may cause partial dissolution along the surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003ec-e\u003c/b\u003e). Many of the Fe particles in the OB were not agglomerated or had only one or two other particles nearby, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003ef-I\u003c/b\u003e with corresponding elemental maps for Fe and O. Near the exogenous Fe particles were also copious amounts of biomineralized iron in the form of ferritin nanoparticles which typically are 3\u0026ndash;12 nm in diameter and do not agglomerate. The ferritin are typically smaller in size compared with the exogenous Fe (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003ef; j, l-m\u003c/b\u003e) and were identified using electron energy loss spectroscopy (EELS) analysis based on their FeL3 and FeL2 edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003ed\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThus, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (magnetite) was detected on the grid post-exposure and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles were likewise found in olfactory bulb in an Fe-exposed brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFe nanoparticles are identified in the olfactory bulb Bottom \u0026ldquo;B\u0026rdquo;: STEM image shows two regions marked with yellow square where Fe particles translocated to the olfactory bulb (a). A magnified view of the agglomerate in (a) is shown in (b) and further magnified in (c)-(e) with corresponding elemental maps for Fe and O distribution. The STEM image in (f) illustrates the location of two isolated exogenous magnetite (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) particles near a corpora amylacea body with copious endogenous Fe particle accumulation \u0026ldquo;ferritin NP\u0026rdquo;. The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e are further magnified in (g)-(i) and show no Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e rims. The ferritin NP are illustrated at higher magnification in the STEM images in (j) and (l) with corresponding elemental map for Fe in (m). ELLS analysis of ferritin NP of the region marked in (j) with a yellow square. Correspondingly, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e was detected in olfactory bulb from an Fe-exposed brain.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBrain Neurotransmitter Levels Two Days Post-Exposure:\u003c/h2\u003e \u003cp\u003eLevels of glutamatergic, serotonergic and dopaminergic classes of neurotransmitters were examined in the striatum and cerebellum two days post termination of exposure.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eStriatum\u003c/span\u003e \u0026ndash; Striatal levels of neurotransmitters in Fe- and air-exposed females (top row) and males (bottom row) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. No significant differences in levels of any of the neurotransmitters within any of the three classes examined were found in response to inhaled Fe in males. In the case of females, however, Fe-based changes were found in both glutamatergic and dopaminergic systems. Specifically, within the glutamatergic system, significant Fe-related increases were found in levels of glutamine (Gln: +19%, F(1,4)\u0026thinsp;=\u0026thinsp;7.88, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048) and glutamate (Glu: +29%, F(1,4)\u0026thinsp;=\u0026thinsp;15.38, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0172), with a marginal increase in levels of GABA (+\u0026thinsp;31%, F(1,4)\u0026thinsp;=\u0026thinsp;4.66, p\u0026thinsp;=\u0026thinsp;0.097). Marginal Fe-related increases were observed in levels of the dopamine metabolites DOPAC (+\u0026thinsp;118%; F(1,4)\u0026thinsp;=\u0026thinsp;6.12, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.069) and HVA (+\u0026thinsp;83%; F(1,4)\u0026thinsp;=\u0026thinsp;6.65, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.061).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eCerebellum\u003c/span\u003e - As in striatum, no significant changes were found in Fe-exposed male cerebellum in glutamatergic, dopaminergic or serotonergic neurotransmitter levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In female Fe-exposed cerebellum, significant increases were seen in levels of serotonin (5HT: +83%, (F(1,4)\u0026thinsp;=\u0026thinsp;7.83, p\u0026thinsp;=\u0026thinsp;0.049) along with marginal increases in the 5HT metabolite 5-HIAA (+\u0026thinsp;97%, (F(1,4)\u0026thinsp;=\u0026thinsp;7.47, p\u0026thinsp;=\u0026thinsp;0.052) within the serotonergic system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMagnetic Resonance Histological Imaging Two Days Post-Exposure:\u003c/h2\u003e \u003cp\u003eMagnetic resonance histological imaging of brains collected within 48 hr of exposure revealed significant changes in mice exposed to Fe.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSex\u0026thinsp;+\u0026thinsp;Exposure\u0026thinsp;+\u0026thinsp;Sex:Exposure Model\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the results of a 2-Way ANOVA with interaction model for diffusion tensor imaging, fractional anisotropy and volumetric changes. This gives a high-level overview of changes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiffusion Tensor Imaging Changes\u003c/b\u003e \u003cb\u003e\u0026ndash;\u003c/b\u003e As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the olfactory bulb was significantly changed in response to Fe, with mean, radial and axial diffusivity contrasts for the sex:exposure interaction. The male and female response was in the opposite directions, with increases in Fe-exposed females in mean, radial and axial diffusivity, and reductions in mean, radial and axial diffusivity in olfactory bulb in Fe-exposed males. Post hoc testing (Tukey-Kramer HSD method via multcompare in MATLAB) indicated that for mean, radial and axial diffusivity, the female Fe versus male Fe was the most significant pairwise comparison set (p\u0026thinsp;\u0026lt;\u0026thinsp;0.1).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSummary of MRI Changes with model of\u003c/b\u003e \u003cb\u003e2-Way ANOVA (Sex and Exposure), Interactions\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDIFFUSION TENSOR IMAGING CHANGES\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStructure\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStatistical Summary, Sex:Exposure\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMALE Air vs Fe\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFEMALE Air vs Fe\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOlfactory Bulb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s F 1.04\u003c/p\u003e \u003cp\u003eRadial: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s F 1.06\u003c/p\u003e \u003cp\u003eAxial: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s F 0.982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean: -5.3%\u003c/p\u003e \u003cp\u003eRadial: -4.9%\u003c/p\u003e \u003cp\u003eAxial: -5.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean: 5.0%\u003c/p\u003e \u003cp\u003eRadial: 4.8%\u003c/p\u003e \u003cp\u003eAxial: 5.3%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFRACTIONAL ANISOTROPY CHANGES\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStructure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eStatistical Summary, Sex:Exposure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMALE Air vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eFEMALE Air vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePosterior Amygdalar Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen\u0026rsquo;s F 0.847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-11.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVOLUMETRIC CHANGES\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStructure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eStatistical Summary, Sex:Exposure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMALE Air vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eFEMALE Air vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGeniculate Group, Dorsal Thalamus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s F 0.948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedial Geniculate Complex, Ventral Part\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen\u0026rsquo;s F 0.919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedial Geniculate Complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen\u0026rsquo;s F 0.851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eP-values included in table are uncorrected. Negative (-) % change signifies that Air is larger and positive (+) % change signifies that Fe is larger. Volumetric findings were controlled by brain volume of each specimen, thus normalized volume.\u003c/p\u003e \u003cp\u003eSex is a significant for volumetric changes in the 2-Way ANOVA Models for these structures: Central Amygdalar Nucleus (Uncorrected p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s F 1.37), Anteroventral Nucleus of Thalamus, (Uncorrected p\u0026thinsp;\u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s F 1.07), Prelimbic Area (Uncorrected p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s F 0.876), Orbital Area (Uncorrected p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen\u0026rsquo;s F 0.888), Superior Colliculus-Sensory Related (Uncorrected p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen\u0026rsquo;s F 0.887), and Ventral Posteromedial Nucleus of the Thalamus (Uncorrected p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 Cohen\u0026rsquo;s F 1.00). There were no structures with significant changes in diffusion tensor metrics or normalized volume related to exposure alone.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFractional Anisotropy\u003c/b\u003e \u0026ndash; Analyses revealed one structure, Posterior Amygdalar Nucleus, that was significantly changed for the sex:exposure interaction. Male mice exhibited a decrease in the fractional anisotropy following Fe exposure (-11.3%), while female mice showed an increase in response to Fe exposure (+\u0026thinsp;5.2%).\u003c/p\u003e \u003cp\u003e \u003cb\u003eVolume Changes\u003c/b\u003e \u0026ndash; There were significant changes in multiple components of the geniculate complex (most child to parent structure ordering: Medial Geniculate Complex, Ventral Part; Medial Geniculate Complex; Geniculate Group, Dorsal Thalamus). Specifically, male mice had increases in the volume (+\u0026thinsp;5.1%, +\u0026thinsp;2.7%, and +\u0026thinsp;3.5%) with Fe exposure, while female mice showed decreases in volume (-6.2%, -3.9%, -2.1%) in response to Fe exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStratified by Sex, Changes due to Exposure\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results of stratified by sex Kruskal Wallis non-parametric ANOVAs indicating significant diffusion tensor imaging, fractional anisotropy and volumetric changes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiffusion Tensor Imaging Changes -\u003c/b\u003e Olfactory bulb was significantly influenced by Fe in both female and male mice, but again in opposite directions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additional analyses revealed sex-dependent changes in olfactory bulb diffusivity consistent with increased myelin damage and axonal loss. Specifically, the mean male response to Fe exposure (axial: 0.348 +/- 0.0171 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e x mm\u003csup\u003e2\u003c/sup\u003e/s, radial: 0.270 +/- 0.0105 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e x mm\u003csup\u003e2\u003c/sup\u003e/s, mean: 0.296 +/- 0.0127 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e x mm\u003csup\u003e2\u003c/sup\u003e/s) was lower than that than of the mean female response to Fe exposure (axial: 0.373 +/- 0.00649 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e x mm\u003csup\u003e2\u003c/sup\u003e/s, radial: 0.286 +/- 0.0.0360 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e x mm\u003csup\u003e2\u003c/sup\u003e/s, mean: 0.315 +/- 0.00441 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e x mm\u003csup\u003e2\u003c/sup\u003e/s), assisting in explaining why in the post hoc analysis of the overall 2-Way ANOVA model, this pairwise comparison was also the most changed, i.e., the most different pairing.\u003c/p\u003e \u003cp\u003eAdditional significant changes in the diffusion tensor metrics were unique to each sex. For male mice, there are significant reductions in olfactory areas (mean: -4.2%, axial \u0026minus;\u0026thinsp;4.8%), and hippocampal commissures (radial: -4.4%). For female mice, there are significant increases in the anterior olfactory nucleus (axial: +5.4%).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFractional Anisotropy\u003c/b\u003e \u0026ndash; Alterations in fractional anisotropy in the MRI assessment were male-specific \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Fe-exposed males showed significant reductions in fractional anisotrophy (FA) in posterior amygdalar nucleus (-11.3%), hypoglossal nucleus (-10.7%), pretectal region (-10.3%), basal amygdalar nucleus (-7.0%), subicular regiona (-6.6%), cortical amygdalar zones (-6.4%), fimbria (-6.1%), cranial nerves (-5.6%), trigeminal nerve (-5.4%), and CA1 (-2.4%). Increases in FA in Fe-exposed males were seen in medial preoptic nucleus (+\u0026thinsp;8.9%), nucleus accumbens (+\u0026thinsp;7.9%), and ventral part of striatum (+\u0026thinsp;4.1%).\u003c/p\u003e \u003cp\u003e \u003cb\u003eVolumetric Changes -\u003c/b\u003e As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, male Fe-exposed mice showed a significant increase in the normalized volume of the substantia nigra compact part (+\u0026thinsp;8.0%), midline group of the dorsal thalamus (+\u0026thinsp;5.2%), the posterior complex of the thalamus (+\u0026thinsp;4.1%), the spinal vestibular nucleus (+\u0026thinsp;3.6%), and the geniculate group, dorsal thalamus (+\u0026thinsp;3.5%). Males concurrently exhibited significant volumetric reductions in the trigeminal nerve (-6.0%), the optic tract and chiasm (-4.7%), epithalamus (-3.0%), and of the bed nuclei of the stria terminalis (-2.1%).\u003c/p\u003e \u003cp\u003eFemales showed a significant 5.5% increase in the normalized volume of the vestibulocerebellar nucleus with corresponding significant reductions in volume which were largest in the ventral medial geniculate complex (-6.2%, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), but also showed significant reductions in the lateral amygdala nucleus (-3.6%) and the sensory related portions of the medulla (-2.4%).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of MRI Changes with model of \u003cem\u003eKruskal Wallis non-parametric ANOVA on Exposure, Stratified by Sex\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDIFFUSION TENSOR IMAGING CHANGES\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMALE\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFEMALE\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStructure\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAir vs Fe\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eStructure\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAir vs Fe\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003eOlfactory Bulb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean: -5.3%\u003c/p\u003e \u003cp\u003eRadial: -4.9%\u003c/p\u003e \u003cp\u003eAxial: -5.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003eOlfactory Bulb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean: 5.0%\u003c/p\u003e \u003cp\u003eRadial: 4.8%\u003c/p\u003e \u003cp\u003eAxial: 5.3%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOlfactory Areas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean: -4.2%\u003c/p\u003e \u003cp\u003eAxial: -4.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnterior Olfactory Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxial: 5.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHippocampal commissures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRadial: -4.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFRACTIONAL ANISOTROPY CHANGES\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMALE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003eFEMALE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStructure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAir vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eStructure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eAir vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003ePosterior Amygdalar Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-11.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypoglossal Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-10.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePretectal Region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-10.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedial Preoptic Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNucleus Accumbens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasomedial Amygdalar Nucelus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubicular Region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCortical Amygdalar Zones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFimbria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCranial Nerves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrigeminal Nerve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStriatum Ventral Region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-2.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVOLUMETRIC CHANGES\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMALE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003eFEMALE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStructure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAir vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eStructure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eAir vs Fe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstantia Nigra Compact Part\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e Medial Geniculate Complex, Ventral Part\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrigeminal Nerve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVestibulocerebellar Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMidline Group of the Dorsal Thalamus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLateral Amygdalar Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptic Tract and Chiasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-4.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedulla Sensory Related\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePosterior Complex of the Thalamus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpinal Vestibular Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e Geniculate group, dorsal thalamus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpithalamus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-3.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBed nuclei of the stria terminalis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-2.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e+ Structures which appeared significant in the 2-Way ANOVA analysis, considering sex and exposure together.\u003c/p\u003e \u003cp\u003eFor all entries, uncorrected P-values is \u0026lt;\u0026thinsp;0.05 and estimated Cohen\u0026rsquo;s F is 1.73. Negative (-) % change signifies that Air is larger and positive (+) % change signifies that Fe is larger. Volumetric findings were controlled by brain volume of each specimen, thus is normalized volume\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAβ42 and Tau 6 Months Post-Exposure:\u003c/h2\u003e \u003cp\u003eAs measured approximately 6 months post exposure to inhaled Fe, neither protein levels of pS199 tau, total tau, and Aβ42 concentrations in the frontal cortex or in hippocampus were affected in male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In contrast, Fe-exposed female mice exhibited significantly higher concentrations in frontal cortex of pS199 tau (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.76, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) and of total tau (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.63, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008), but similar concentrations of Aβ42 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.99, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.35) as compared to air-exposed controls. Fe-exposed female mice also exhibited significantly higher hippocampal concentrations of total tau (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.57, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) than did air-exposed controls, but showed similar concentrations of hippocampal pS199 tau (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.50, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.17) and Aβ42 (\u003cem\u003et\u003c/em\u003e = -1.23, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.25).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral Changes Post Exposure:\u003c/h2\u003e \u003cp\u003e \u003cb\u003eLocomotor Activity Levels -\u003c/b\u003e Fe-exposed males showed no significant differences from air-exposed males in any measures of locomotor activity levels: ambulatory distance, ambulatory episode, ambulatory time, jump counts, jump time, rest time, stereotypic counts, stereotypic time, vertical counts, or vertical time (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Similarly, females, regardless of treatment group, performed equivalently across all measures of locomotor behavior.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNovel Object Recognition -\u003c/b\u003e In session 1 of NOR, mice, regardless of sex and treatment, spent equivalent amounts of time with both the left- and the right-placed object as determined by comparing time spent with left or right object vs. half of total interaction time (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), confirming an absence of spatial or activity level bias that could influence results in session 2 determination of the NOR recognition index. Recognition index in session 2, calculated as time spent with the novel object divided by total time spent with both objects averaged 58% and 68%, respectively, for male and female air control mice, consistent with recognition of a novel stimulus. This recognition index was not influenced by Fe exposure in male mice. However, Fe-exposed female mice displayed a 31% significantly lower recognition index than their air-exposed controls (\u003cem\u003et\u003c/em\u003e = -5.19, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRadial Arm Maze -\u003c/b\u003e Radial arm maze performance was assessed over 3 sessions in females and 5 sessions in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). No significant effects of Fe on percent errors were seen in male mice over the course of the 5 sessions of testing, with both groups showing chance levels of accuracy in session 1 and slight declines thereafter. In contrast, while chance levels of errors were also seen in female mice in sessions 1 and 2, levels of errors in female control mice dropped in session 3 by almost 30%, whereas no such change was found in Fe-exposed females, resulting in a marginally significant interaction in the repeated measures analysis (time x treatment (F(2, 9)\u0026thinsp;=\u0026thinsp;0.060), with a significant day 3 reduction confirmed in a subsequent post-hoc t-test (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.86, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017). Notably, levels of percent errors in Session 3 in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, \u003cb\u003ebottom\u003c/b\u003e), including both air- and Fe-exposed mice, were significantly correlated with hippocampal levels of phosphorylated tau (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.37; F (1,11)\u0026thinsp;=\u0026thinsp;6.44, p\u0026thinsp;=\u0026thinsp;0.0275).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePost Behavior Neurotransmitter Changes:\u003c/h2\u003e \u003cp\u003eChanges in striatal and cerebellar neurotransmitter levels were examined post behavioral testing in mice that underwent behavioral assessments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStriatum\u003c/b\u003e \u0026ndash; In contrast to effects seen 48 hr post exposure, significant Fe-induced changes in striatal neurotransmitters post behavioral testing were seen only in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). In Fe-exposed females, the only change seen was a marginal (8%) increase in excitotoxicity (glutamate/GABA; F(1,10)\u0026thinsp;=\u0026thinsp;3.29, p\u0026thinsp;=\u0026thinsp;0.0998). In contrast, changes in all three classes of neurotransmitters were now evident in males. Effects within the class of glutamatergic neurotransmitters included a significant 19% increase in levels of GABA (F(1,10)\u0026thinsp;=\u0026thinsp;6.25, p\u0026thinsp;=\u0026thinsp;0.031), a 40% marginal increase in levels of glutamine (F(1,10)\u0026thinsp;=\u0026thinsp;4.59, p\u0026thinsp;=\u0026thinsp;0.058) and a 22% marginal increase in glutamate (F(1,10)\u0026thinsp;=\u0026thinsp;4.4, p\u0026thinsp;=\u0026thinsp;0.062). Changes seen within the class of serotonergic neurotransmitters in Fe-exposed male striatum included an 84% marginal increase in kynurenine (F(1,10)\u0026thinsp;=\u0026thinsp;4.71, p\u0026thinsp;=\u0026thinsp;0.0552), as well as a 32% significant reduction in levels of serotonin (F(1,10)\u0026thinsp;=\u0026thinsp;18.32, p\u0026thinsp;=\u0026thinsp;0.0016). Change also occurred in response to Fe within the class of dopaminergic neurotransmitters, specifically in reduced dopamine turnover, with a 44% significant reduction in the ratio of HVA/DA (F(1,10)\u0026thinsp;=\u0026thinsp;5.2, p\u0026thinsp;=\u0026thinsp;0.046) as well as a significant 25% reduction in the ratio of DOPAC/DA (F(1,10)\u0026thinsp;=\u0026thinsp;5.4, p\u0026thinsp;=\u0026thinsp;0.043).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCerebellum\u003c/b\u003e - Post behavior changes in cerebellar neurotransmitter function (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e) were more limited. In females, Fe-induced changes were limited to a marginal 17% increase in levels of glutathione (F(1,12)\u0026thinsp;=\u0026thinsp;4.1, p\u0026thinsp;=\u0026thinsp;0.066). In males exposed to Fe, a significant 28% increase in levels of serotonin turnover were observed (F(1,12)\u0026thinsp;=\u0026thinsp;5.69, p\u0026thinsp;=\u0026thinsp;0.035) in conjunction with a marginal 20% increase in dopamine turnover (DOPAC/DA; F(1,12)\u0026thinsp;=\u0026thinsp;3.52, p\u0026thinsp;=\u0026thinsp;0.085).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTrans-Sulfuration Markers\u003c/h2\u003e \u003cp\u003eChanges in levels of markers within the trans-sulfuration pathway, specifically methionine, homo-cysteine, cysteine and glutathione, were measured in both striatum and cerebellum at 2 days post exposure (labeled Pre) and after behavioral testing (labeled Post) and are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e. In striatum, females evidenced significant increases in glutathione even at 2 days post Fe exposure (GSH: +23%, F(1,4)\u0026thinsp;=\u0026thinsp;10.17, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0332), with levels remaining elevated albeit not significantly when measured post behavioral testing. Notably, males showed a marked 44% significant increase in glutathione (F(1,10)\u0026thinsp;=\u0026thinsp;6.65, p\u0026thinsp;=\u0026thinsp;0.028) but this was not evident until post behavioral testing, and was accompanied by a significant 31% increase in levels of cysteine (F(1,10)\u0026thinsp;=\u0026thinsp;10.07, p\u0026thinsp;=\u0026thinsp;0.0099). Some evidence of a delayed increase in glutathione was also seen in females in cerebellum (F(1,12)\u0026thinsp;=\u0026thinsp;4.098, p\u0026thinsp;=\u0026thinsp;0.066).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBased on the accumulating evidence linking both AP exposure [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and elevated brain Fe [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] concentrations with risk for neurodegenerative diseases and disorders, the current study sought to examine in a mouse model the hypothesis that inhaled Fe, as would occur through AP exposures, would reach brain and would reproduce features of such diseases and disorders, and do so in a sex-dependent manner. Consistent with these hypotheses, speciation analyses of TEM grids from the exposure chambers confirmed the presence of spark-generated, exogenous magnetite which was likewise seen in olfactory bulb, the region that would be the initial port of entry into brain following nasal olfactory uptake in response to Fe inhalation, confirming the uptake of exogenous Fe. Additionally, characteristics of neurodegenerative diseases and disorders occurred in response to Fe inhalation and differed notably by sex. Specifically, in females, numerous outcomes characteristics of AD were seen, including increased levels of phosphorylated tau in frontal cortex and total tau in both frontal cortex and hippocampus, and increases in olfactory bulb diffusivity potentially indicative of myelin damage and/or axonal loss. Fe-exposed females also exhibited impaired memory, as assessed using both using a novel object recognition paradigm and a radial arm maze paradigm, with the latter impairments significantly correlated with levels of frontal cortical total tau. In contrast, the profile of consequences in males differed notably and showed characteristics associated with PD that included increases in volume of the substantia nigra pars compacta concurrently with reductions in the volume of the trigeminal nerve, in addition to reductions in mean, radial and axial diffusivity in olfactory bulb and hippocampus and altered fractional anisotropy changes in multiple subcortical structures.\u003c/p\u003e \u003cp\u003eS/TEM analysis coupled with EDS confirmed the presence of magnetite in mouse olfactory bulb following Fe inhalation. These exogenous Fe nanoparticles can be identified based on their structural and crystalline similarity to the spark generated Fe nanoparticles produced and collected on TEM grids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Such findings are consistent with nasal olfactory uptake of elemental AP particles and salts in both rats and humans [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e99\u003c/span\u003e], including Fe, Mn, Cd, Ni, Hg, Al, Co, Zn, and Cu [99, 100, 101, 102, 103, 104], through translocation across olfactory epithelium by olfactory neuronal cells along neuronal tracts, followed by transportation into olfactory bulb, and movement to other brain regions [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Sensory nerves in the upper and lower respiratory tract can also translocate particles [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e106\u003c/span\u003e] that reach e.g., the trigeminal ganglion or the vagal nerve [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Such Increases in Fe have been reported in brain and nerves of mice in other studies after inhalation exposures of Fe nanoparticles [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e107\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe olfactory route of exposure/uptake of Fe is of particular interest to reports defining the staging of neurodegenerative disorders, including AD and PD, as olfactory bulb is found as an early site of change in both [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. In the current study, Fe-based changes in olfactory bulb diffusivity were found in both sexes, and appeared as reductions in diffusivity in males, but as increases in diffusivity in females. While it is not yet clear how such changes translate into neuropathological features, such findings could suggest potential myelin abnormalities and/or axonal changes [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. Interestingly, in our prior studies of developmental air pollution exposures, hypermyelination of the corpus callosum occurred in both sexes following gestational exposures that was correlated with Fe inclusions in corpus callosum in females and axonal changes including thick myelin sheaths with \u0026ldquo;holes\u0026rdquo; indicative of damage [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e110\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e111\u003c/span\u003e], while postnatal exposures resulted in a male-biased hypomyelination [\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e112\u003c/span\u003e]. Future research is needed to associate these AP related myelin changes with MR diffusivity measures. In terms of disease staging, Braak staging of PD suggests a pathogen entering the brain via the nasal route or trigeminal or via the vagal nerve as initial sites of pathology [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e113\u003c/span\u003e], consistent with nanoparticle routes to brain, with studies reporting olfactory bulb as an area that accumulates alpha-synuclein aggregates, a hallmark of PD. Of additional support is a study that examined metal concentrations and distributions in the human olfactory bulb in PD and found elevated Fe in the PD olfactory bulb [\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. In the case of AD, the unfolded protein response that leads to upregulation of beta-amyloid and tau production, hallmark features of AD, is found throughout the olfactory system even in early-stage Braak pathological staging of AD, including Braak stages 0 and 1 [\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e115\u003c/span\u003e, \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e116\u003c/span\u003e]. The olfactory bulb pathology indicated by increased diffusivity in MRI analyses in female Fe-exposed mice is consistent with the well documented involvement of olfactory bulb in dementia and AD, and indicative of changes to white matter microstructure. In accord with these findings, olfactory loss is considered to be a component of the long prodromal phase of AD as well as in PD [\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e117\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFemales showed several additional characteristic features of AD following Fe inhalation, including volumetric reductions in regions likewise implicated in AD and include structural connections with olfactory system [\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e118\u003c/span\u003e]. For example, reductions in amygdala volume have long been recognized in AD [\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e119\u003c/span\u003e] and have been found to be of greater magnitude in lateral amygdala [\u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e120\u003c/span\u003e], the specific nucleus in which volumetric reductions were seen in Fe-exposed female brain in this study. It is notable that hippocampus and amygdala show early involvement in AD and receive projections from the olfactory bulb [\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e121\u003c/span\u003e]. In contrast to these regions, increases in size of the vestibulocerebellar nucleus (flocculus) were concurrently found in female Fe-exposed brains. While prior studies have cited involvement of cerebellum in AD, these were seen as reductions [\u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e122\u003c/span\u003e, \u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e123\u003c/span\u003e] as determined in individuals with diagnosed dementia. One potential basis for this increase could be an early compensatory plasticity. While compensatory mechanisms in AD have been proposed [\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e124\u003c/span\u003e], the earliest changes in AD and possible compensatory processes have yet to be established. As with some other outcomes, studies of the time course of the consequences of Fe would provide important mechanistic information.\u003c/p\u003e \u003cp\u003eFemales exposed to Fe likewise showed evidence of impaired memory, with these impairments correlated with increased levels of phosphorylated tau in hippocampus. A hallmark of AD is the neuropathological misfolding and aggregation of two brain proteins, amyloid β (Aβ) and tau. Correlations between elevation of brain Fe and formation of neurofibrillary tangles have been reported [\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e125\u003c/span\u003e]. Fe interacts with tau: Fe\u003csup\u003e3+\u003c/sup\u003e can induce aggregation of hyperphosphorylated tau, while reduction of Fe\u003csup\u003e3+\u003c/sup\u003e to Fe\u003csup\u003e2+\u003c/sup\u003e reverses the effect, as shown in hyperphosphorylated tau obtained from AD brains [\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e125\u003c/span\u003e, \u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e126\u003c/span\u003e]. The reduction of Fe\u003csup\u003e3+\u003c/sup\u003e to Fe\u003csup\u003e2+\u003c/sup\u003e can solubilize iron since Fe\u003csup\u003e2+\u003c/sup\u003e can be present and transported in ionic form and hence be mobilized. Another way to reduce Fe\u003csup\u003e3+\u003c/sup\u003e to Fe\u003csup\u003e2+\u003c/sup\u003e is to reduce the common Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e (hematite) to Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (magnetite) which does not mobilize or remove Fe from the tissue regions. The correlation of hippocampal phosphorylated tau levels with impaired memory is of particular interest given the reported role of tau in cognitive deficits in AD [\u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e127\u003c/span\u003e, \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e128\u003c/span\u003e]. It has been suggested that entorhinal cortex tau accumulation underlies hippocampal activation and memory loss over time [\u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e129\u003c/span\u003e], while cerebrospinal levels of tau have been found to be predictive of reductions in hippocampal volume and interpreted as reflecting neuronal loss [\u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e130\u003c/span\u003e]. In AD, significant amounts of Fe are found within Aβ plaques and tau-based neurofibrillary proteins [\u003cspan citationid=\"CR132\" class=\"CitationRef\"\u003e131\u003c/span\u003e, \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e132\u003c/span\u003e, \u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e133\u003c/span\u003e]. Although Fe promotes Aβ aggregation, and binds to the Aβ peptide with binding affinity increasing Aβ aggregation, further potentiating Aβ neurotoxicity [\u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e134\u003c/span\u003e, \u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e135\u003c/span\u003e, \u003cspan citationid=\"CR137\" class=\"CitationRef\"\u003e136\u003c/span\u003e, \u003cspan citationid=\"CR138\" class=\"CitationRef\"\u003e137\u003c/span\u003e], changes in Aβ were not found in these studies. It is possible that Aβ pathology needs to be evaluated in a transgenic mouse line with relevance to human AD protein production or requires more protracted exposures.\u003c/p\u003e \u003cp\u003eA distinct effect found in males included a significant increase in volume of the substantia nigra pars compacta, the site of dopamine cell loss leading to motor dysfunction in male-dominated PD. The increase seen in substantia nigra volume is notable, given reports of enlarged substantia nigra hyperechogenicity that have been shown to correlate with Fe accumulation in substantia nigra [\u003cspan citationid=\"CR139\" class=\"CitationRef\"\u003e138\u003c/span\u003e], and constitute a predictive risk for PD [\u003cspan citationid=\"CR140\" class=\"CitationRef\"\u003e139\u003c/span\u003e], a male-biased disorder resulting from loss of dopamine neuronal cell bodies in this region. Notably, reductions in striatal dopamine turnover were observed concurrently. The increase in substantia nigra pars compacta volume seen here in males, again, could reflect an early compensatory response to Fe inhalation [\u003cspan citationid=\"CR141\" class=\"CitationRef\"\u003e140\u003c/span\u003e] and emphasizes the need to further understand compensatory responses particularly during the early trajectory of this disease. Additional studies are also needed to assess the relationship of hyperechogenicity to changes assessed by neuroimaging, as efforts to date have not been conclusive [\u003cspan citationid=\"CR142\" class=\"CitationRef\"\u003e141\u003c/span\u003e]. In addition, assessment of potential neuropathology in this region could further define the meaning of the increased volume. Males likewise showed changes in fractional anisotropy values for a range of brain structures, most of which involved reductions, including nuclei of the amygdala, the hypoglossal nucleus, the pretectal region, and the trigeminal nerve, all of which are ultimately directly or indirectly interconnected via the trigeminal nerve. The reductions in FA could suggest the potential for demyelination, inflammation, edema and axonal loss. Moreover, these findings are consistent with a significant reduction in volume of the trigeminal nerve that was also observed in males.\u003c/p\u003e \u003cp\u003eOther studies of Fe inhalation suggest potential sources of the deficits seen here. For example, Fe inhalation was found to increase numbers of activated microglial cells and levels of Il-1β in olfactory bulb of adult female mice after a 6 hr/day, 5 days/week for 5 week exposure to 200 \u0026micro;g/m\u003csup\u003e3\u003c/sup\u003e iron-soot inhalation that included 40 \u0026micro;g/m\u003csup\u003e3\u003c/sup\u003e of Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. Inhaled Fe nanoparticles have also been reported to produce focal damage to the myelin sheath of a nerve fiber in the olfactory bulb [\u003cspan citationid=\"CR143\" class=\"CitationRef\"\u003e142\u003c/span\u003e], consistent with the fact that Fe is requisite for myelination [\u003cspan citationid=\"CR144\" class=\"CitationRef\"\u003e143\u003c/span\u003e]. Interestingly, no effects on brain connectivity were observed in either Fe exposed male or female brains. Future studies are needed to evaluate if a more protracted exposure or prolonged aging would reveal effects of Fe inhalation on the neural connectome.\u003c/p\u003e \u003cp\u003eIncreased oxidative stress is another feature seen in neurodegenerative diseases including AD [\u003cspan citationid=\"CR145\" class=\"CitationRef\"\u003e144\u003c/span\u003e] and PD [\u003cspan citationid=\"CR146\" class=\"CitationRef\"\u003e145\u003c/span\u003e] as caused by impairments in antioxidant capacity [\u003cspan citationid=\"CR147\" class=\"CitationRef\"\u003e146\u003c/span\u003e, \u003cspan citationid=\"CR148\" class=\"CitationRef\"\u003e147\u003c/span\u003e]. Evidence for such alterations was seen in both sexes. In the case of females, this included increases in striatal glutathione even at 48 hr post exposure, but not seen post-behavioral testing. Typically, reductions in glutathione have been associated with mild cognitive impairment and cognitive decline in AD [\u003cspan citationid=\"CR145\" class=\"CitationRef\"\u003e144\u003c/span\u003e, \u003cspan citationid=\"CR149\" class=\"CitationRef\"\u003e148\u003c/span\u003e] that are considered to promote Aβ deposition and tau phosphorylation [\u003cspan citationid=\"CR150\" class=\"CitationRef\"\u003e149\u003c/span\u003e] and lead to apoptosis [\u003cspan citationid=\"CR151\" class=\"CitationRef\"\u003e150\u003c/span\u003e]. In the current study, the increase measured in females shortly after the end of the exposure period could represent an adaptive or compensatory response. Correspondingly, in a study of Fe exposure in SH-SY5Y cells, a biphasic GSH response occurred with increases followed by decreases, as Fe exposure concentration increased [\u003cspan citationid=\"CR152\" class=\"CitationRef\"\u003e151\u003c/span\u003e]. Of further note, however, were the concurrent increases in female striatal levels of glutamate and its precursor, glutamine, both of which are involved in ferroptotic processes, a form of cell death arising from Fe accumulation and consequent oxidative stress and lipid peroxidation. Specifically, glutamate inhibits cystine uptake by the cystine/glutamate antiporter requisite to the production of glutathione [\u003cspan citationid=\"CR153\" class=\"CitationRef\"\u003e152\u003c/span\u003e]. In the case of males, glutathione increases were also seen, but not until post-behavioral testing. In conjunction with the differences in outcomes of Fe inhalation by sex, the differences in timing of the increases in glutathione suggest the potential for sex differences in antioxidant response/timing that likely contributes to the sex differences in neurodegenerative disease prevalence/phenotypes. Such differences are consistent with known sex differences in redox homeostasis in brain [\u003cspan citationid=\"CR154\" class=\"CitationRef\"\u003e153\u003c/span\u003e]. Clearly, additional markers of Fe-induced oxidative stress and ferroptosis will be required to determine the extant mechanisms underlying these changes and their inter-relationships.\u003c/p\u003e \u003cp\u003eThe sex-related differences in response to Fe seen in this study are consistent with a significant literature documenting sex differences in Fe handling and response [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR155\" class=\"CitationRef\"\u003e154\u003c/span\u003e, \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e155\u003c/span\u003e]. Of particular relevance to the issue of critical periods of exposure, our prior studies have shown that male brain appeared to be more sensitive to inhaled Fe than female brain when such exposures were carried out developmentally in conjunction with exposures to SO\u003csub\u003e2\u003c/sub\u003e, another component of AP, which has been shown to enhance the uptake of Fe into the central nervous system [\u003cspan citationid=\"CR157\" class=\"CitationRef\"\u003e156\u003c/span\u003e]. Under those conditions, effects of inhalational exposures of C57Bl/6J mice to much lower levels of Fe nanoparticles (1 ug/m\u003csup\u003e3\u003c/sup\u003e) in conjunction with SO\u003csub\u003e2\u003c/sub\u003e (500 ppb from postnatal days 4\u0026ndash;7 and 10\u0026ndash;13 (human third trimester brain equivalent; [\u003cspan citationid=\"CR158\" class=\"CitationRef\"\u003e157\u003c/span\u003e]) for 4 hr/day, were particularly dramatic in males, and included a marked brain metal dyshomeostasis in frontal cortex along with striatal excitatory:inhibitory (glutamate) imbalance and marked increases in levels of dopamine and metabolites, concurrently with reductions in serotonin and metabolites at postnatal day 14 [\u003cspan citationid=\"CR159\" class=\"CitationRef\"\u003e158\u003c/span\u003e]. Consequently, in addition to sex, timing of exposure and potential cumulative exposures, the chemical speciation of the Fe in air pollution is an additional modifier of neurotoxicity.\u003c/p\u003e \u003cp\u003eAs previously noted, AP that includes Fe as a contaminant represent a lifelong exposure. Whether early exposures result in developmental reprogramming of brain systems with long term consequences or whether and how the profile of effects observed in the current study would change with more protracted or more cumulative Fe inhalation exposures are all as yet unknown. Findings from the current study underscore the need for defining vulnerable periods of exposure, and assessment of both cumulative exposures and of the longitudinal trajectory of Fe-related brain impacts, as well as the mechanisms that contribute to sex and brain region differences in vulnerability to inhaled Fe.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe current study demonstrates that inhaled exogenous Fe UFPs and reach brain, where, in female brain it can produce features characteristic of AD, while in male brain it can alter volume of regions involved in PD. Levels of UFPs, considered the most reactive component of AP, are not regulated by the United States Environmental Protection Agency given monitoring complications and based, in part, on the assumption that UFP concentrations would decline with regulated reductions in levels of PM\u003csub\u003e2.5\u003c/sub\u003e. However, this assumption has not necessarily proven to be the case, which in fact, can be inversely related [\u003cspan citationid=\"CR160\" class=\"CitationRef\"\u003e159\u003c/span\u003e, \u003cspan citationid=\"CR161\" class=\"CitationRef\"\u003e160\u003c/span\u003e, \u003cspan citationid=\"CR162\" class=\"CitationRef\"\u003e161\u003c/span\u003e, \u003cspan citationid=\"CR163\" class=\"CitationRef\"\u003e162\u003c/span\u003e, \u003cspan citationid=\"CR164\" class=\"CitationRef\"\u003e163\u003c/span\u003e]. Findings from the current study underscore the need to further understand the potential neurotoxic consequences of metal constituents within UFPs, especially Fe as it is an essential, redox active metal that has already known to be elevated in brain in numerous neurodegenerative diseases. Further studies of inhaled Fe could yield information ultimately critical to understanding mechanisms of neurodegeneration. Additionally, evidence from the current studies suggests that regulation of Fe levels in AP, and, in particular, in areas of high concentrations such as subways, might provide public health protection against a broad set of neurodegenerative diseases and disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and Consent to Participation -\u003c/strong\u003e This study was carried out in accordance with relevant guidelines and regulations. All mice used were treated according to protocols approved by the University of Rochester Medical Center Institutional Animal Care and Use Committee and Committee on Animal Resources (approval #102208/2010-046E) and in accordance with NIH guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication -\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests -\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding -\u003c/h2\u003e \u003cp\u003eSupported by NIH grants R01 ES032260 (Cory-Slechta, PI), R35 ES031689-01A1 (Cory-Slechta, PI) and P30 ES001247 (Lawrence, PI).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDAC-S, MS and GO designed the study and prepared the manuscript; DC and RG carried out and monitored all exposures; JVG, AM, KW KC and EM carried out measurements of outcome variables; UG carried out Fe speciation analyses in TEM grids and olfactory bulb; J.A.G. and K.X carried out the magnetic resonance histology and diffusivity analyses. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the University of Rochester Electron Microscopy Shared Resource Laboratory for their assistance in processing tissue for Electron Microscopy and Spectroscopy techniques.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials -\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed in the current study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaringer SL, Simpson IA, Connor JR. Brain iron acquisition: An overview of homeostatic regulation and disease dysregulation. 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Atmospheric Environment. 2001;35 25:4357-66; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1352-2310(01)00229-1\u003c/span\u003e\u003cspan address=\"10.1016/S1352-2310(01)00229-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. ://WOS:000170749200014.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"particle-and-fibre-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pftx","sideBox":"Learn more about [Particle and Fibre Toxicology](http://particleandfibretoxicology.biomedcentral.com)","snPcode":"12989","submissionUrl":"https://submission.nature.com/new-submission/12989/3","title":"Particle and Fibre Toxicology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"iron, air pollution, tau, olfactory bulb, memory, Alzheimer’s disease, Parkinson’s disease, substantia nigra","lastPublishedDoi":"10.21203/rs.3.rs-5314480/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5314480/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBoth excess brain Fe and air pollution (AP) exposures are associated with increased risk for multiple neurodegenerative disorders. Fe is a redox-active metal that is abundant in AP from traffic and industrial sources and in U.S. subway systems. Exposures to AP and associated contaminants, such as Fe, are lifelong and could therefore contribute to elevated brain Fe observed in neurodegenerative diseases, particularly via nasal olfactory uptake of ultrafine particle AP. These studies tested the hypotheses that exogenously generated Fe oxide nanoparticles could reach brain following inhalational exposures and produce neurotoxic effects consistent with neurodegenerative diseases and disorders in adult C57/Bl6J mice exposed by inhalation to Fe nanoparticles at a concentration similar to those found in underground subway systems (~\u0026thinsp;150 ug/m\u003csup\u003e3\u003c/sup\u003e) for 20 days. Olfactory bulb sections and exposure chamber TEM grids were analyzed for Fe speciation. Measures included brain volumetric and diffusivity changes, levels of striatal and cerebellar neurotransmitters and trans-sulfuration markers, quantification of frontal cortical and hippocampal Aβ42, total tau and phosphorylated tau and behavioral alterations in locomotor activity and memory.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eParticle speciation confirmed similarity of Fe oxides (mostly magnetite) found on chamber TEM grids and in olfactory bulb. Alzheimer\u0026rsquo;s disease (AD) like characteristics were seen in Fe-exposed females including increased olfactory bulb diffusivity, impaired memory and increased accumulation of total and phosphorylated tau, with total hippocampal tau levels significantly correlated with increased errors in the radial arm maze. Fe-exposed males showed increased volume of the substantia nigra pars compacta, a region critical to the motor impairments seen in Parkinson\u0026rsquo;s disease (PD), in conjunction with reduced volume of the trigeminal nerve and optic tract and chiasm.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eInhaled Fe oxide nanoparticles appeared to lead to olfactory bulb uptake. Further, these exposures reproduced characteristic features of neurodegenerative diseases in a sex-dependent manner, with females evidencing features similar to those seen in AD and effects in regions in males associated with PD. As such, prolonged inhaled Fe exposure via AP may be a risk factor for neurodegenerative diseases, and regulation of air Fe levels in enclosed areas like subway stations may have broad public health protective effects. More research is needed to improve our translation from these rodent studies to human exposures. We suggest, prolonged inhaled FE exposure via AP is a suggested risk factor for neurodegenerative changes in mice and given the similarities of these changes to changes observed in human AD, these data may have broad public health protections effects.\u003c/p\u003e","manuscriptTitle":"Brain Iron Accumulation in Neurodegenerative Disorders: Does Air Pollution Play a Role?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 17:11:54","doi":"10.21203/rs.3.rs-5314480/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-25T13:09:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-25T12:08:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-23T01:59:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Particle and Fibre Toxicology","date":"2024-10-22T22:05:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"particle-and-fibre-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pftx","sideBox":"Learn more about [Particle and Fibre Toxicology](http://particleandfibretoxicology.biomedcentral.com)","snPcode":"12989","submissionUrl":"https://submission.nature.com/new-submission/12989/3","title":"Particle and Fibre Toxicology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9fb4636-26c2-485a-ba78-fd2823348cf7","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T15:59:30+00:00","versionOfRecord":{"articleIdentity":"rs-5314480","link":"https://doi.org/10.1186/s12989-025-00622-z","journal":{"identity":"particle-and-fibre-toxicology","isVorOnly":false,"title":"Particle and Fibre Toxicology"},"publishedOn":"2025-05-01 15:56:54","publishedOnDateReadable":"May 1st, 2025"},"versionCreatedAt":"2024-11-05 17:11:54","video":"","vorDoi":"10.1186/s12989-025-00622-z","vorDoiUrl":"https://doi.org/10.1186/s12989-025-00622-z","workflowStages":[]},"version":"v1","identity":"rs-5314480","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5314480","identity":"rs-5314480","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}