HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity.

Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild-type (WT) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R2 relaxation rate within the substantia nigra of the paraquat-treated mice compared to their saline-treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R2 relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L-ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT. Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease modifier for Parkinson's disease. Moreover, the finding that the HFE mutant mice are resistant to paraquat may provide a model in which to study resistant mechanisms to neurotoxicants.

Default mode network deactivation during odor-visual association.

Default mode network (DMN) deactivation has been shown to be functionally relevant for goal-directed cognition. In this study, the DMN's role during olfactory processing was investigated using two complementary functional magnetic resonance imaging (fMRI) paradigms with identical timing, visual-cue stimulation, and response monitoring protocols. Twenty-nine healthy, non-smoking, right-handed adults (mean age = 26 ± 4 years, 16 females) completed an odor-visual association fMRI paradigm that had two alternating odor + visual and visual-only trial conditions. During odor + visual trials, a visual cue was presented simultaneously with an odor, while during visual-only trial conditions the same visual cue was presented alone. Eighteen of the twenty-nine participants (mean age = 27.0 ± 6.0 years, 11 females) also took part in a control no-odor fMRI paradigm that consisted of a visual-only trial condition which was identical to the visual-only trials in the odor-visual association paradigm. Independent Component Analysis (ICA), extended unified structural equation modeling (euSEM), and psychophysiological interaction (PPI) were used to investigate the interplay between the DMN and olfactory network. In the odor-visual association paradigm, DMN deactivation was evoked by both the odor + visual and visual-only trial conditions. In contrast, the visual-only trials in the no-odor paradigm did not evoke consistent DMN deactivation. In the odor-visual association paradigm, the euSEM and PPI analyses identified a directed connectivity between the DMN and olfactory network which was significantly different between odor + visual and visual-only trial conditions. The results support a strong interaction between the DMN and olfactory network and highlights the DMN's role in task-evoked brain activity and behavioral responses during olfactory processing. Hum Brain Mapp 38:1125-1139, 2017. © 2016 Wiley Periodicals, Inc.

The relationship between iron dyshomeostasis and amyloidogenesis in Alzheimer's disease: Two sides of the same coin.

The dysregulation of iron metabolism in Alzheimer's disease is not accounted for in the current framework of the amyloid cascade hypothesis. Accumulating evidence suggests that impaired iron homeostasis is an early event in Alzheimer's disease progression. Iron dyshomeostasis leads to a loss of function in several enzymes requiring iron as a cofactor, the formation of toxic oxidative species, and the elevated production of beta-amyloid proteins. Several common genetic polymorphisms that cause increased iron levels and dyshomeostasis have been associated with Alzheimer's disease but the pathoetiology is not well understood. A full picture is necessary to explain how heterogeneous circumstances lead to iron loading and amyloid deposition. There is evidence to support a causative interplay between the concerted loss of iron homeostasis and amyloid plaque formation. We hypothesize that iron misregulation and beta-amyloid plaque pathology are synergistic in the process of neurodegeneration and ultimately cause a downward cascade of events that spiral into the manifestation of Alzheimer's disease. In this review, we amalgamate recent findings of brain iron metabolism in healthy versus Alzheimer's disease brains and consider unique mechanisms of iron transport in different brain cells as well as how disturbances in iron regulation lead to disease etiology and propagate Alzheimer's pathology.

The effect of iron in MRI and transverse relaxation of amyloid-beta plaques in Alzheimer's disease.

Dysregulation of neural iron is known to occur during the progression of Alzheimer's disease. The visualization of amyloid-beta (Aß) plaques with MRI has largely been credited to rapid proton relaxation in the vicinity of plaques as a result of focal iron deposition. The goal of this work was to determine the relationship between local relaxation and related focal iron content associated with Aß plaques. Alzheimer's disease (n=5) and control tissue (n=3) sample slices from the entorhinal cortex were treated overnight with the iron chelator deferoxamine or saline, and microscopic gradient-echo MRI datasets were taken. Subsequent to imaging, the same slices were stained for Aß and iron, and then compared with regard to parametric R2 * relaxation maps and gradient-echo-weighted MR images. Aß plaques in both chelated and unchelated tissue generated MR hypo-intensities and showed relaxation rates significantly greater than the surrounding tissue. The transverse relaxation rate associated with amyloid plaques was determined not to be solely a result of iron load, as much of the relaxation associated with Aß plaques remained following iron chelation. The data indicate a dual relaxation mechanism associated with Aß plaques, such that iron and plaque composition synergistically produce transverse relaxation.

Histological-MRI correlation in the primary motor cortex of patients with amyotrophic lateral sclerosis.

PURPOSE: To establish the relationship between ALS histopathology and quantitative MRI metrics. MATERIALS AND METHODS: ALS patients (N = 8) in advanced stages of the disease were enrolled and, immediately after death, the brain of each patient was removed. Freshly excised ALS tissue was imaged at 3.0 Tesla with T1 and T2 mapping protocols and subsequently stained with astrocyte, myelin, and neuronal markers. Measures of ALS histological stains were compared with the internal control (primary visual cortex) and longitudinal parametric maps. RESULTS: Post-mortem T1 -weighted images demonstrate diminished contrast between gray and white matter and alterations in T1 relaxation within the primary motor cortex. An increase in astrocyte number and reactivity as well as evident neuronal loss, a decrease in axonal density, and unraveling of the myelin sheaths in subcortical white matter were found in the ALS primary motor cortex exhibiting significant T1 relaxation and contrast changes. CONCLUSION: This study provides a histopathological basis for differences in MR T1 contrast and relaxation seen in the ALS brain.

[Met5]-enkephalin preserves diffusion metrics in EAE mice.

Multiple sclerosis is a chronic progressive neurological disorder that has few distinctive biomarkers associated with disease progression or response to therapy. This research investigated whether non-invasive imaging correlated with animal behavior and morphological indicators of disease in response to serum levels of [Met5]-enkephalin. Using the experimental autoimmune encephalomyelitis (EAE) model, adult female C57BL/6 J mice were randomized to receive daily injections of 0.1 mg/kg naltrexone (NTX) (= low dose naltrexone, LDN), 10 mg/kg Opioid Growth Factor (OGF) (chemically termed [Met5]-enkephalin) or saline beginning at the time of disease induction. Daily composite behavior scores were recorded over a 30-day period based on tail tone, gait, righting reflex, and limb strength. Prior to disease onset (day 7), and at peak disease (day 18), mice were imaged and tissues (blood and spinal cord) collected at day 30 for serum analyses of OGF and morphology. Serum OGF levels of EAE mice treated with saline were significantly reduced from baseline and from normal mice. Longitudinal cohort data demonstrated an increase in fractional anisotropy in all cohorts by day 18. There was a significant decrease in radial diffusivity in the saline group seen at day 18 whereas the axial diffusivity was not altered amongst treatment groups. Treatment with OGF or LDN resulted in mean diffusivity rates that were comparable to baseline (normal) levels at days 7 and 18. Luxol fast blue staining of the lumbar spinal cords demonstrated a 16 % reduction in myelin staining in saline treated EAE animals when compared to OGF and LDN treated EAE mice. Immunohistochemistry with Olig2 (pan-oligodendrocyte marker) and myelin basic protein (MBP) revealed that OGF and LDN treatment restored the area (%) of MBP and number of oligodendrocytes to that of normal spinal cord (∼75 %). Saline treated EAE mice had more demyelination and fewer oligodendrocytes than normal mice. Collectively, these data suggest that a panel of biomarkers including imaging, serum biomarker levels, and behavior correlate with progression of disease, and may begin to validate use of specific non-invasive markers for MS.

Dietary lipophilic iron accelerates regional brain iron-load in C57BL6 mice.

Impaired brain iron homeostatic mechanisms, independent of pathological hallmarks, are harmful to the brain because excess free iron can cause DNA, protein, and lipid damage via oxidative stress. The goal of this study was to evaluate the longitudinal effect of chronic iron overload and deficiency in the vertebrate brain. Ten-week-old C57BL6 male mice were randomly assigned to one of four unique dietary regiments for 1 year: iron-deficient, normal iron, and two different concentrations of lipophilic iron diet containing 3,5,5-trimethylhexanoyl ferrocene (TMHF). Longitudinal MRI parametrics were used to assess the location and extent of ferric iron distribution. Tissue collected at 12 months was used to directly measure iron-load, protein alterations, and histological metrics. While the iron-deficient diet did not alter brain iron stores, 0.11% TMHF and early exposure with 0.5% TMHF elevated brain iron by roughly 40 and 100%, respectively. R 2 rate increased more in the TMHF groups within iron rich brain regions. Increased brain iron concentration was linearly correlated with an increase in L-ferritin expression, and TMHF diet was found to increase L-ferritin within the olfactory bulb, neocortex, pallidum, thalamus, corpus callosum, CA3 regions of the hippocampus, and substantia nigra. Moreover, gliosis and oxidative stress were detected in the TMHF groups in the regions associated with iron-load. Spatial memory impairment was evident in the iron-loaded mice. This work illustrates that lipophilic iron elevates brain iron in a regionally specific fashion and positions dietary TMHF administration as a model for brain iron overloading.

Reduced Cerebral White Matter Integrity Assessed by DTI in Cognitively Normal H63D-HFE Polymorphism Carriers.

BACKGROUND AND PURPOSE: The H63D-HFE single nucleotide polymorphism (SNP) has been associated with brain iron dysregulation; however, the emergent role of this missense variant in brain structure and function has yet to be determined. Previous work has demonstrated that HFE SNP carriers have reduced white matter magnetic resonance imaging (MRI) proton relaxation rates. The mechanism by which white matter alterations perturb MRI relaxation is unknown as is how these metrics are related to myelin integrity. METHODS: Fifteen subjects heterozygous for the HFE-H63D SNP and 25 controls with wild-type HFE had diffusion-weighted, anatomical MRIs taken, and underwent cognitive assessment. Fractional anisotropy (FA), mean diffusion (MD), and mode of anisotropy (MO) were calculated from the diffusion dataset to investigate the relationship between the H63D-HFE SNP and myelin integrity. RESULTS: A decrease in FA, an increase in MD, and an increase in MO are demonstrated in multiple H63D-HFE polymorphism carrier white matter tracts. Regions with altered diffusion metrics are notably located in heavily myelinated white matter association fibers, such as the anterior corona radiata and longitudinal fasciculi. CONCLUSIONS: The MRI data presented here demonstrate that H63D-HFE polymorphism carriers have diffusivity changes in white matter compared to wild-type subjects. The reduced integrity white matter tracts in H63D-HFE carriers are hypothesized to be related to increased susceptibility of these late-myelinating regions to cellular stress induced by oligodendrocyte iron dyshomeostasis.

Dietary lipophilic iron alters amyloidogenesis and microglial morphology in Alzheimer's disease knock-in APP mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized pathologically by amyloid beta (Aß) deposition, microgliosis, and iron dyshomeostasis. Increased labile iron due to homeostatic dysregulation is believed to facilitate amyloidogenesis. Free iron is incorporated into aggregating amyloid peptides during Aß plaque formation and increases potential for oxidative stress surrounding plaques. The goal of this work was to observe how brain iron levels temporally influence Aß plaque formation, plaque iron concentration, and microgliosis. We fed humanized APPNL-F and APPNL-G-F knock-in mice lipophilic iron compound 3,5,5-trimethylhexanoyl ferrocene (TMHF) and iron deficient diets for twelve months. TMHF elevated brain iron by 22% and iron deficiency decreased brain iron 21% relative to control diet. Increasing brain iron with TMHF accelerated plaque formation, increased Aß staining, and increased senile morphology of amyloid plaques. Increased brain iron was associated with increased plaque-iron loading and microglial iron inclusions. TMHF decreased IBA1+ microglia branch length while increasing roundness indicative of microglial activation. This body of work suggests that increasing mouse brain iron with TMHF potentiates a more human-like Alzheimer's disease phenotype with iron integration into Aß plaques and associated microgliosis.

Gamma Knife radiosurgery of saccular aneurysms in a rabbit model.

OBJECTIVEIntracranial aneurysms are vascular abnormalities associated with neurological morbidity and mortality due to risk of rupture. In addition, many aneurysm treatments have associated risk profiles that can preclude the prophylactic treatment of asymptomatic lesions. Gamma Knife radiosurgery (GKRS) is a standard treatment for trigeminal neuralgia, tumors, and arteriovenous malformations. Aneurysms associated with arteriovenous malformations have been noted to resolve after treatment of the malformation. The aim of this study was to determine the efficacy of GKRS treatment in a saccular aneurysm animal model.METHODSAneurysms were surgically produced using an elastase-induced aneurysm model in the right common carotid artery of 10 New Zealand white rabbits. Following initial observation for 4 years, each rabbit aneurysm was treated with a conformal GKRS isodose of 25 Gy to the 50% margin. Longitudinal MRI studies obtained over 2 years and terminal measures obtained at multiple time points were used to track aneurysm size and shape index modifications.RESULTSAneurysms did not rupture or involute during the observation period. Whole aneurysm and blood volume averages decreased with a linear trend, at rates of 1.7% and 1.6% per month, respectively, over 24 months. Aneurysm wall percent volume increased linearly at a rate of 0.3% per month, indicating a relative thickening of the aneurysm wall during occlusion. Nonsphericity of the average volume, aspect ratio, and isoperimetric ratio of whole aneurysm volume all remained constant. Histopathological samples demonstrated progressive reduction in aneurysm size and wall thickening, with subintimal fibrosis. Consistent shape indices demonstrate stable aneurysm patency and maintenance of minimal rupture risk following treatment.CONCLUSIONSThe data indicate that GKRS targeted to saccular aneurysms is associated with histopathological changes and linear reduction of aneurysm size over time. The results suggest that GKRS may be a viable, minimally invasive treatment option for intracranial aneurysm obliteration.

Reduced white matter MRI transverse relaxation rate in cognitively normal H63D-HFE human carriers and H67D-HFE mice.

Mutations within the HFE protein gene sequence have been associated with increased risk of developing a number of neurodegenerative disorders. To this effect, an animal model has been created which incorporates the mouse homologue to the human H63D-HFE mutation: the H67D-HFE knock-in mouse. These mice exhibit alterations in iron management proteins, have increased neuronal oxidative stress, and a disruption in cholesterol regulation. However, it remains undetermined how these differences translate to human H63D carriers in regards to white matter (WM) integrity. To this endeavor, MRI transverse relaxation rate (R2) parametrics were employed to test the hypothesis that WM alterations are present in H63D human carriers and are recapitulated in the H67D mice. H63D carriers exhibit widespread reductions in brain R2 compared to non-carriers within white matter association fibers in the brain. Similar R2 decreases within white matter tracts were observed in the H67D mouse brain. Additionally, an exacerbation of age-related R2 decrease is found in the H67D animal model in white matter regions of interest. The decrease in R2 within white matter tracts of both species is speculated to be multifaceted. The R2 changes are hypothesized to be due to alterations in axonal biochemical tissue composition. The R2 changes observed in both the human-H63D and mouse-H67D data suggest that modified white matter myelination is occurring in subjects with HFE mutations, potentially increasing vulnerability to neurodegenerative disorders.

Cortical iron regulation and inflammatory response in Alzheimer's disease and APPSWE/PS1ΔE9 mice: a histological perspective.

Disruption of iron homeostasis and increased glial response are known to occur in brains afflicted by Alzheimer's disease (AD). While the APP/PS1 transgenic mouse model recapitulates the hallmark amyloid-beta plaque pathology of AD, it does so in a different neuronal mileu than humans. Understanding the iron characteristics and glial response of the APP/PS1 model is important when testing new treatment procedures and translating these results. Brain tissue from AD patients, APP/PS1 mice, and controls were stained for iron, H- and L-ferritin, microglia, astrocytes, Aß40∕42, and degenerating neurons. The histological data demonstrate differences in ferritin, iron distribution, gliosis, and Aß plaque composition between APP/PS1 and AD tissue. Specifically, an association between focal iron deposition and Aß plaques is found ubiquitously throughout the AD tissue and is not observed in the APP/PS1 mouse model. Ferritin, microglia, and astrocyte staining show differential response patterns to amyloid plaques in AD and the APP/PS1 tissue. Aß 40 and 42 antibody and thioflavin staining demonstrate morphological differences in plaque composition. The histological data support the hypothesis that iron distribution, iron management, and glial response histologically differ between the APP/PS1 and AD brain. Acknowledging the caveat that there are distinct plaque, iron, and glial contrasts between the AD brain and the APP/PS1 mouse is crucial when utilizing this model.

Olfactory deficit detected by fMRI in early Alzheimer's disease.

Alzheimer's disease (AD) is accompanied by smell dysfunction, as measured by psychophysical tests. Currently, it is unknown whether AD-related alterations in central olfactory system neural activity, as measured by functional magnetic resonance imaging (fMRI), are detectable beyond those observed in healthy elderly. Moreover, it is not known whether such changes are correlated with indices of odor perception and dementia. To investigate these issues, 12 early stage AD patients and 13 nondemented controls underwent fMRI while being exposed to each of three concentrations of lavender oil odorant. All participants were administered the University of Pennsylvania Smell Identification Test (UPSIT), the Mini-Mental State Examination (MMSE), the Mattis Dementia Rating Scale-2 (DRS-2), and the Clinical Dementia Rating Scale (CDR). The blood oxygen level-dependent (BOLD) signal at primary olfactory cortex (POC) was weaker in AD than in HC subjects. At the lowest odorant concentration, the BOLD signals within POC, hippocampus, and insula were significantly correlated with UPSIT, MMSE, DRS-2, and CDR scores. The BOLD signal intensity and activation volume within the POC increased significantly as a function of odorant concentration in the AD group, but not in the control group. These findings demonstrate that olfactory fMRI is sensitive to the AD-related olfactory and cognitive functional decline.

MRI and histological analysis of beta-amyloid plaques in both human Alzheimer's disease and APP/PS1 transgenic mice.

PURPOSE: To investigate the relationship between MR image contrast associated with beta-amyloid (Abeta) plaques and their histology and compare the histopathological basis of image contrast and the relaxation mechanism associated with Abeta plaques in human Alzheimer's disease (AD) and transgenic APP/PS1 mouse tissues. MATERIALS AND METHODS: With the aid of the previously developed histological coil, T(2) (*)-weighted images and R(2) (*) parametric maps were directly compared with histology stains acquired from the same set of Alzheimer's and APP/PS1 tissue slices. RESULTS: The electron microscopy and histology images revealed significant differences in plaque morphology and associated iron concentration between AD and transgenic APP/PS1 mice tissue samples. For AD tissues, T(2) (*) contrast of Abeta-plaques was directly associated with the gradation of iron concentration. Plaques with significantly less iron load in the APP/PS1 animal tissues are equally conspicuous as the human plaques in the MR images. CONCLUSION: These data suggest a duality in the relaxation mechanism where both high focal iron concentration and highly compact fibrillar beta-amyloid masses cause rapid proton transverse magnetization decay. For human tissues, the former mechanism is likely the dominant source of R(2) (*) relaxation; for APP/PS1 animals, the latter is likely the major cause of increased transverse proton relaxation rate in Abeta plaques. The data presented are essential for understanding the histopathological underpinning of MRI measurement associated with Abeta plaques in humans and animals.

Direct magnetic resonance imaging of histological tissue samples at 3.0T.

Direct imaging of a histological slice is challenging. The vast difference in dimension between planar size and the thickness of histology slices would require an RF coil to produce a uniform RF magnetic (B1) field in a 2D plane with minimal thickness. In this work a novel RF coil designed specifically for imaging a histology slice was developed and tested. The experimental data demonstrate that the coil is highly sensitive and capable of producing a uniform B1 field distribution in a planar region of histological slides, allowing for the acquisition of high-resolution T2 images and T2 maps from a 60-microm-thick histological sample. The image intensity and T2 distributions were directly compared with histological staining of the relative iron concentration of the same slice. This work demonstrates the feasibility of using a microimaging histological coil to image thin slices of pathologically diseased tissue to obtain a precise one-to-one comparison between stained tissue and MR images.