Regular articleActivated iron-containing microglia in the human hippocampus identified by magnetic resonance imaging in Alzheimer disease
Introduction
Alzheimer disease (AD) is pathologically characterized primarily by the presence of amyloid beta (Aβ) plaques and tau-rich neurofibrillary tangles in the clinical context of memory impairment (Braak and Braak, 1991, Selkoe, 2008). Although Aβ has a strong genetic link to AD (Tanzi, 2012), the mechanism of plaque-mediated neurodegeneration is unclear because of the presence of plaques in healthy elderly controls (Arnold et al., 2013, Davis et al., 1999) and the lack of a strong correlation between plaque deposition and the severity of dementia (Terry et al., 1991). Neurofibrillary tangles have a strong correlation with mnemonic impairment, but the genetics of tau mutations are associated with other neurodegenerative disorders (Goedert and Jakes, 2005), and the mechanism of tau pathogenesis and relationship to Aβ is unclear (Spires-Jones and Hyman, 2014). In comparison to amyloid plaques, soluble Aβ oligomers have a stronger correlation with clinical complaints (Tomic et al., 2009). Aβ in vitro has been shown to convert the redox-inactive ferric iron (Fe3+), normally present in the storage form of iron (ferrihydrite), to the redox-active ferrous (Fe2+) iron, a catalyst of the Fenton reaction that generates toxic free radicals, a driver of inflammation (Everett et al., 2014a, Everett et al., 2014b). This in vitro result is relevant because abnormal iron accumulation occurs in AD (Lovell et al., 1998, Smith et al., 1997), and rather than being mineralized as the typical storage form of Fe3+ within ferrihydrite, it is more composed of magnetite and wüstite, which both possess Fe2+ (Collingwood et al., 2008, Pankhurst et al., 2008, Quintana et al., 2004). The broader neuroinflammation literature in AD has shown colocalization of microglia (the inflammatory cells of the nervous system) with sites of AD pathology including the medial temporal lobe (Sheng et al., 1995) and colocalization of ferritin (which stores intracellular iron) with microglia in AD (Connor et al., 1992, Grundke-Iqbal et al., 1990). A genome-wide expression study found a 10-fold gain-of-function in AD of microglial gene networks, and this gene network had the strongest correlation of all gene modules with AD neuropathology (Zhang et al., 2013). Microglial cell-surface antigen CD33 expression is elevated in AD and inversely correlated with plaque clearance (Gandy and Heppner, 2013, Griciuc et al., 2013). A murine model of AD incorporating an altered immune-redox state recapitulates the pathological progression of amyloid, tau, and neuronal loss seen in humans better than other mouse models (Colton et al., 2014). Thus, iron-associated microglial-driven neuroinflammation may be a significant driver behind neuronal destruction, synergistic with amyloid (Gallagher et al., 2012, McGeer and McGeer, 2013). However, no study to date has quantified or even identified the distribution of iron-containing microglia in the medial temporal lobe or described the relationship of these microglia to both amyloid and tau deposition.
High-field magnetic resonance imaging (MRI) is exquisitely sensitive to microscopic iron and can be performed at ultra-high resolution ex vivo. However, all ex vivo MRI work in AD has focused exclusively on Aβ plaques. Although initial efforts at plaque visualization reported contradictory results (Benveniste et al., 1999, Dhenain et al., 2002), later work using murine and human specimens definitively visualized iron associated with plaques in the entorhinal cortex (the hippocampus was not specifically depicted and microglia not assessed) (Meadowcroft et al., 2009).
In this study, we hypothesize that foci of hypointense signal on MR in AD would reflect iron deposits, and these deposits would be within activated microglia rather than Aβ plaques, reflective of an inflammatory component of AD. We imaged 5 AD and 5 control human hippocampal specimens with 7T MRI at ultra-high resolution (0.1 mm isotropic) using 2 iron-sensitive pulse sequences. To discern the histological correlate underlying MR hypointensity, we sectioned the same specimens and performed successive triple staining using DAB (3,3’-diaminobenzidine tetrahydrochloride) enhanced Perl's stain for iron, CD 163 for microglia, and an immunostain for Aβ. These stains were separable in the same section by color subtraction and coregistered with each other and the MRI. We additionally employed phospho-tau-AT8 immunostaining on adjacent sections to explore the relationship with neurofibrillary pathology. Finally, we delineated the exact location of these MR hypointense foci with regard to hippocampal subfields by performing and coregistering anatomic Nissl, myelin, and acetylcholinesterase (AChE) stains.
Section snippets
Overview of methodology
Five 4-mm thick slabs from fixed human AD and control specimens (Table 1) were placed in a container and supported by thin plastic sheets (Fig. 1A). The container fit tightly within a solenoidal coil to minimize the distance between the coil and the tissue, thereby optimizing signal sensitivity and image quality (Fig. 1B and C). Slabs were then paraffin-embedded and sectioned at 10-μm thickness in their entirety. Every 30th section was double-stained with DAB-iron and CD163, and the entire
Specimen MRI
Scanning of the medial temporal lobe at 100-μm isotropic resolution with 7T MRI demonstrated exquisite anatomic detail, clearly showing the microanatomy and lamination of the hippocampus similar to other efforts with specimen MRI (Fig. 1, Fig. 4, Fig. 5) (Adler et al., 2014, Augustinack et al., 2014).
Alzheimer MR microscopy
Four of 5 medial temporal lobe AD specimens were at the level of the hippocampal body: these 4 demonstrated numerous very dark and tiny signal voids concentrated primarily in the subiculum,
Discussion
Using ultra-high field 7T ex vivo MRI of AD, we identified focal hypointensities located in the hippocampus in AD, specifically primarily within the subiculum, a novel imaging finding that could be specific for AD. Coregistration of the MRI with the histology suggested that these foci represent iron deposits that may largely be within microglia, also known as siderophages. This finding was neuropathologically present in concentrated form in most of the AD cases, and not within the control
Disclosure statement
Dr. Michael Zeineh and Dr. Brian Rutt both receive research funding from General Electric Healthcare. The authors have no other conflicts of interest to disclose.
Acknowledgements
The authors thank Dr. Karl Zilles and Dr. Nicola Palomero-Gallagher for their detailed assistance in segmenting the prosubiculum, Dr. Edward Plowey and Dr. Michelle James for their comments on this manuscript, Dr. Jarrett Rosenberg for his advice on statistical analyses, Jason Parnes for his assistance developing the slide scanning procedure, and Dr. Scott Atlas for his comments on MRI-histology interpretation. Dr. Zeineh received an RSNA Research Fellow Award that funded a portion of this
References (50)
- et al.
Histology-derived volumetric annotation of the human hippocampal subfields in postmortem MRI
Neuroimage
(2014) - et al.
High field magnetic resonance microscopy of the human hippocampus in Alzheimer's disease: quantitative imaging and correlation with iron
Neuroimage
(2012) - et al.
Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer's disease
Neurobiol. Aging
(2013) - et al.
MRI parcellation of ex vivo medial temporal lobe
Neuroimage
(2014) - et al.
A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain
Neuroimage
(2011) - et al.
Different beta-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity
Neurobiol. Aging
(2012) - et al.
Propagation of tau pathology in a model of early Alzheimer's disease
Neuron
(2012) - et al.
Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: possible relation to oxidative stress
J. Neurol. Sci.
(1996) - et al.
Microglia, amyloid, and cognition in Alzheimer's disease: an [11C](R)PK11195-PET and [11C]PIB-PET study
Neurobiol. Dis.
(2008) - et al.
Microglia as dynamic and essential components of the amyloid hypothesis
Neuron
(2013)
Mutations causing neurodegenerative tauopathies
Biochim. Biophys. Acta
Alzheimer's disease risk gene CD33 inhibits microglial uptake of amyloid beta
Neuron
Copper, iron and zinc in Alzheimer's disease senile plaques
J. Neurol. Sci.
Study of the localization of iron, ferritin, and hemosiderin in Alzheimer's disease hippocampus by analytical microscopy at the subcellular level
J. Struct. Biol.
Electron nanodiffraction and high-resolution electron microscopy studies of the structure and composition of physiological and pathological ferritin
J. Struct. Biol.
Biochemistry and Molecular Biology of Amyloid β-Protein and the Mechanism of Alzheimer's Disease
Handb Clin Neurol.
The intersection of amyloid beta and tau at synapses in Alzheimer's disease
Neuron
Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction
Neurobiol. Dis.
Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer's disease
Cell
Detection of neuritic plaques in Alzheimer's disease by magnetic resonance microscopy
Proc. Natl. Acad. Sci. U. S. A.
Neuropathological stageing of Alzheimer-related changes
Acta Neuropathol.
Early clinical PET imaging results with the novel PHF-tau radioligand [F-18]-T807
J. Alzheimers Dis.
Three-dimensional tomographic imaging and characterization of iron compounds within Alzheimer's plaque core material
J. Alzheimers Dis.
mNos2 deletion and human NOS2 replacement in Alzheimer disease models
J. Neuropathol. Exp. Neurol.
A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains
J. Neurosci. Res.
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2023, Bioorganic ChemistryCitation Excerpt :These studies suggested that neuroinflammatory factors may stimulate iron overload by regulating the expression of iron transporters, followed by a positive feedback effect that aggravates neuroinflammation in AD brains. Microglia are closely related to iron overload, and iron-positive microglia are more abundant than astrocytes in AD brains [208–209]. Iron plays a direct role in Aβ-stimulated neuroinflammation.