Effects of Alzheimer’s disease and formalin fixation on the different mineralised-iron forms in the human brain

Abstract

Iron accumulation in the brain is a phenomenon common to many neurodegenerative diseases, perhaps most notably Alzheimer’s disease (AD). We present here magnetic analyses of post-mortem brain tissue of patients who had severe Alzheimer’s disease, and compare the results with those from healthy controls. Isothermal remanent magnetization experiments were performed to assess the extent to which different magnetic carriers are affected by AD pathology and formalin fixation. While Alzheimer’s brain material did not show higher levels of magnetite/maghemite nanoparticles than corresponding controls, the ferrihydrite mineral, known to be found within the core of ferritin proteins and hemosiderin aggregates, almost doubled in concentration in patients with Alzheimer’s pathology, strengthening the conclusions of our previous studies. As part of this study, we also investigated the effects of sample preparation, by performing experiments on frozen tissue as well as tissue which had been fixed in formalin for a period of 5 months. Our results showed that the two different preparations did not critically affect the concentration of magnetic carriers in brain tissue, as observable by SQUID magnetometry.

Quantification of different iron forms in the aceruloplasminemia brain to explore iron-related neurodegeneration

“…The concentrations of ferrihydrite-iron in the basal ganglia of our patient with aceruloplasminemia, could not be appropriately compared with results that had been previously obtained from the basal ganglia of healthy subjects and patients with neuroferritinopathy (29), due to substantial methodological differences between studies. The amount of ferrihydrite-iron in the temporal cortex of aceruloplasminemia, though, was six times higher than in healthy subjects and almost four times higher compared to patients with advanced Alzheimer’s disease that were measured under identical conditions (18, 34). Although these comparisons are limited to the temporal cortex, which is on average three times less rich in iron than the deep gray matter structures in aceruloplasminemia, they illustrates once more the severity of iron overload in aceruloplasminemia.…”

“…Absolute SIRM values detected at 100 K in aceruloplasminemia were approximately two orders of magnitude higher than those reported in healthy subjects, at room temperature (41), which suggests that also maghemite-iron levels may have increased as a result of iron overload in aceruloplasminemia. In addition, the concentration of maghemite-iron detected in the temporal cortex of aceruloplasminemia was almost twice that of healthy controls and advanced cases of Alzheimer’s disease that were measured under identical conditions (18, 34). In contrast to ferrihydrite-iron, the regional distribution of maghemite-iron in the aceruloplasminemia brain was not substantially different from that of healthy controls (41), with the cerebellar and brain stem nuclei being relatively rich in this magnetic carrier, although thalamic structures were not specifically investigated by previous studies (41).…”

“…Since formalin fixation is known to reduce total tissue iron content and may have a detrimental effect on protein conformations (44-47), it is likely that the presented absolute concentrations of Fe 3+ ions, ferrihydrite-iron and maghemite-iron in the aceruloplasminemia brain are underestimated compared to in vivo . Therefore, future studies should aim to minimize the duration of formalin fixation (34), and ideally include both aceruloplasminemia and control brain tissue to enable more in-depth analyses of the exact relationship between the different molecular forms of iron that are present in healthy and severely diseased states. On the other hand, the spatial distribution of the different iron forms, as presented in our work, is not significantly affected by prolonged formalin fixation (41).…”

Quantification of different iron forms in the aceruloplasminemia brain to explore iron-related neurodegeneration

“…The concentrations of ferrihydrite-iron in the basal ganglia of our patient with aceruloplasminemia could not be appropriately compared with results that had been previously obtained from the basal ganglia of healthy subjects and patients with neuroferritinopathy ( Hautot et al, 2007 ), due to substantial methodological differences between studies. The amount of ferrihydrite-iron in the temporal cortex of aceruloplasminemia, though, was six times higher than in healthy subjects and almost four times higher compared to patients with advanced Alzheimer’s disease that were measured under identical conditions, on average ( Bulk et al, 2018 , van der Weerd et al, 2020 ). Although these comparisons are limited to the temporal cortex, which is on average three times less rich in iron than the deep gray matter structures in aceruloplasminemia, they illustrate once more the severity of iron overload in the disease.…”

The essential elements of Alzheimer’s disease

“…Reports of elevated postmortem tissue iron are inconsistent. Insufficient detection limits, small sample sizes in early studies, inaccuracies in the clinicopathological diagnosis of AD, and iron-depletion by fixatives may have contributed to this variance ( 88 , 253 , 254 ). A large recent study described that iron accumulation in the inferior temporal cortex could only be found in subjects both diagnosed clinically for AD and confirmed postmortem by standardized criteria ( 255 ).…”