Elsevier

Neurobiology of Aging

Volume 36, Issue 9, September 2015, Pages 2483-2500
Neurobiology of Aging

Regular article
Activated iron-containing microglia in the human hippocampus identified by magnetic resonance imaging in Alzheimer disease

https://doi.org/10.1016/j.neurobiolaging.2015.05.022Get rights and content

Abstract

Although amyloid plaques and neurofibrillary pathology play important roles in Alzheimer disease (AD), our understanding of AD is incomplete, and the contribution of microglia and iron to neurodegeneration is unknown. High-field magnetic resonance imaging (MRI) is exquisitely sensitive to microscopic iron. To explore iron-associated neuroinflammatory AD pathology, we studied AD and control human brain specimens by (1) performing ultra-high resolution ex vivo 7 Tesla MRI, (2) coregistering the MRI with successive histologic staining for iron, microglia, amyloid beta, and tau, and (3) quantifying the relationship between magnetic resonance signal intensity and histological staining. In AD, we identified numerous small MR hypointensities primarily within the subiculum that were best explained by the combination of microscopic iron and activated microglia (p = 0.025), in contradistinction to the relatively lesser contribution of tau or amyloid. Neuropathologically, this suggests that microglial-mediated neurodegeneration may occur in the hippocampal formation in AD and is detectable by ultra-high resolution MRI.

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

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