The coarse-grained plaque: a divergent Aß plaque-type in early-onset Alzheimer's disease.

Alzheimer's disease (AD) is characterized by amyloid-beta (Aß) deposits, which come in myriad morphologies with varying clinical relevance. Previously, we observed an atypical Aß deposit, referred to as the coarse-grained plaque. In this study, we evaluate the plaque's association with clinical disease and perform in-depth immunohistochemical and morphological characterization. The coarse-grained plaque, a relatively large (Ø ≈ 80 µm) deposit, characterized as having multiple cores and Aß-devoid pores, was prominent in the neocortex. The plaque was semi-quantitatively scored in the middle frontal gyrus of Aß-positive cases (n = 74), including non-demented cases (n = 15), early-onset (EO)AD (n = 38), and late-onset (LO)AD cases (n = 21). The coarse-grained plaque was only observed in cases with clinical dementia and more frequently present in EOAD compared to LOAD. This plaque was associated with a homozygous APOE ε4 status and cerebral amyloid angiopathy (CAA). In-depth characterization was done by studying the coarse-grained plaque's neuritic component (pTau, APP, PrPC), Aß isoform composition (Aß40, Aß42, AßN3pE, pSer8Aß), its neuroinflammatory component (C4b, CD68, MHC-II, GFAP), and its vascular attribution (laminin, collagen IV, norrin). The plaque was compared to the classic cored plaque, cotton wool plaque, and CAA. Similar to CAA but different from classic cored plaques, the coarse-grained plaque was predominantly composed of Aß40. Furthermore, the coarse-grained plaque was distinctly associated with both intense neuroinflammation and vascular (capillary) pathology. Confocal laser scanning microscopy (CLSM) and 3D analysis revealed for most coarse-grained plaques a particular Aß40 shell structure and a direct relation with vessels. Based on its morphological and biochemical characteristics, we conclude that the coarse-grained plaque is a divergent Aß plaque-type associated with EOAD. Differences in Aß processing and aggregation, neuroinflammatory response, and vascular clearance may presumably underlie the difference between coarse-grained plaques and other Aß deposits. Disentangling specific Aß deposits between AD subgroups may be important in the search for disease-mechanistic-based therapies.

MRI artifacts in human brain tissue after prolonged formalin storage.

For the interpretation of magnetic resonance imaging (MRI) abnormalities in brain pathology, often ex vivo tissue is used. The purpose of this study was to determine the pathological substrate of several distinct forms of MR hypointensities that were found in formalin-fixed brain tissue with amyloid-beta deposits. Samples of brain cortex were scanned using effective transverse relaxation time-weighted protocols at several resolutions on a 9.4 T MRI scanner. High resolution MRI showed large coarse hypointensities throughout the cortical gray and white matter, corresponding to macroscopic discolorations and microscopic circumscribed areas of granular basophilic neuropil changes, without any further specific tissue reactions or amyloid-beta related pathology. These coarse MRI hypointensities were identified as localized areas of absent neuropil replaced by membrane/myelin sheath remnants using electron microscopy. Interestingly, the presence/absence of these tissue alterations was not related to amyloid deposits, but strongly correlated to the fixation time of the samples in unrefreshed formalin. These findings show that prolonged storaged of formalin fixed brain tissue results in subtle histology artifacts, which show on MRI as hypointensities that on first appearance are indistinguishable from genuine brain pathology. This indicates that postmortem MRI should be interpreted with caution, especially if the history of tissue preservation is not fully known.

High-field MRI of single histological slices using an inductively coupled, self-resonant microcoil: application to ex vivo samples of patients with Alzheimer's disease.

A simple inductively coupled microcoil has been designed to image tissue samples placed on a microscope slide, samples which can subsequently be stained histologically. As the exact same tissue is used for MRI and histology, the two data sets can be compared without the need for complicated image registration techniques. The design can be integrated into any MRI system using existing commercial hardware. Compared with a commercial 25-mm-diameter birdcage, the signal-to-noise ratio was increased by a factor of 3.8, corresponding to an approximate 15-fold reduction in the data acquisition time. An example is shown of ex vivo samples from patients with Alzheimer's disease, in which the coregistration of highly sensitive iron staining and amyloid-ß deposits is confirmed.

MLPAinter for MLPA interpretation: an integrated approach for the analysis, visualisation and data management of Multiplex Ligation-dependent Probe Amplification.

BACKGROUND: Multiplex Ligation-Dependent Probe Amplification (MLPA) is an application that can be used for the detection of multiple chromosomal aberrations in a single experiment. In one reaction, up to 50 different genomic sequences can be analysed. For a reliable work-flow, tools are needed for administrative support, data management, normalisation, visualisation, reporting and interpretation. RESULTS: Here, we developed a data management system, MLPAInter for MLPA interpretation, that is windows executable and has a stand-alone database for monitoring and interpreting the MLPA data stream that is generated from the experimental setup to analysis, quality control and visualisation. A statistical approach is applied for the normalisation and analysis of large series of MLPA traces, making use of multiple control samples and internal controls. CONCLUSIONS: MLPAinter visualises MLPA data in plots with information about sample replicates, normalisation settings, and sample characteristics. This integrated approach helps in the automated handling of large series of MLPA data and guarantees a quick and streamlined dataflow from the beginning of an experiment to an authorised report.

Cerebral amyloidosis: postmortem detection with human 7.0-T MR imaging system.

PURPOSE: To explore the ability of whole-body 7.0-T magnetic resonance (MR) imaging to depict differences in aspects of the cerebral cortex of postmortem human brain specimens with cerebral amyloid beta deposition in connection with Alzheimer disease (AD), Down syndrome, or sporadic or hereditary cerebral amyloid angiopathy (CAA) and control brain specimens lacking such deposition. MATERIALS AND METHODS: This study was approved by the local institutional review board. In all cases, informed consent was obtained to perform autopsy and to use the tissues for research purposes. T2- and T2*-weighted MR imaging was performed in formalin-fixed samples of brain tissue from six subjects with AD changes, seven with CAA, and five subjects without immunohistochemical evidence of cerebral amyloid beta deposition. All MR images were visually assessed for hypointense foci in and inhomogeneity of the cortex. Sensitivity, specificity, and kappa values of these MR imaging features in the detection of histologic changes were calculated. RESULTS: High-spatial-resolution 0.3 x 0.3 x 0.3-mm three-dimensional T2*-weighted images revealed hypointense foci, inhomogeneity of the cortex, or both in all specimens with brain amyloid beta deposition. These MR imaging features were observed in none of the control specimens. CONCLUSION: The finding of postmortem susceptibility-weighted changes in the cerebral cortex of patients with cerebral amyloidosis with a human 7.0-T MR imaging system opens up the possibility of obtaining in vivo radiologic evidence of cerebral amyloid beta deposition.

Postmortem T2*- Weighted MRI Imaging of Cortical Iron Reflects Severity of Alzheimer's Disease.

The value of iron-based MRI changes for the diagnosis and staging of Alzheimer's disease (AD) depends on an association between cortical iron accumulation and AD pathology. Therefore, this study determined the cortical distribution pattern of MRI contrast changes in cortical regions selected based on the known distribution pattern of tau pathology and investigated whether MRI contrast changes reflect the underlying AD pathology in the different lobes. T2*-weighted MRI was performed on postmortem cortical tissue of controls, late-onset AD (LOAD), and early-onset AD (EOAD) followed by histology and correlation analyses. Combining ex vivo high-resolution MRI and histopathology revealed that: 1) LOAD and EOAD have a different distribution pattern of AD pathological hallmarks and MRI contrast changes over the cortex, with EOAD showing more severe MRI changes; 2) per lobe, severity of AD pathological hallmarks correlates with iron accumulation, and hence with MRI. Therefore, iron-sensitive MRI sequences allow detection of the cortical distribution pattern of AD pathology ex vivo.

TGFß pathway deregulation and abnormal phospho-SMAD2/3 staining in hereditary cerebral hemorrhage with amyloidosis-Dutch type.

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) pathology, caused by the E22Q mutation in the amyloid ß (Aß) peptide. Transforming growth factor ß1 (TGFß1) is a key player in vascular fibrosis and in the formation of angiopathic vessels in transgenic mice. Therefore, we investigated whether the TGFß pathway is involved in HCHWA-D pathogenesis in human postmortem brain tissue from frontal and occipital lobes. Components of the TGFß pathway were analyzed with quantitative RT-PCR. TGFß1 and TGFß Receptor 2 (TGFBR2) gene expression levels were significantly increased in HCHWA-D in comparison to the controls, in both frontal and occipital lobes. TGFß-induced pro-fibrotic target genes were also upregulated. We further assessed pathway activation by detecting phospho-SMAD2/3 (pSMAD2/3), a direct TGFß down-stream signaling mediator, using immunohistochemistry. We found abnormal pSMAD2/3 granular deposits specifically on HCHWA-D angiopathic frontal and occipital vessels. We graded pSMAD2/3 accumulation in angiopathic vessels and found a positive correlation with the CAA load independent of the brain area. We also observed pSMAD2/3 granules in a halo surrounding occipital vessels, which was specific for HCHWA-D. The result of this study indicates an upregulation of TGFß1 in HCHWA-D, as was found previously in AD with CAA pathology. We discuss the possible origins and implications of the TGFß pathway deregulation in the microvasculature in HCHWA-D. These findings identify the TGFß pathway as a potential biomarker of disease progression and a possible target of therapeutic intervention in HCHWA-D.

Postmortem MRI and histology demonstrate differential iron accumulation and cortical myelin organization in early- and late-onset Alzheimer's disease.

Previous MRI studies reported cortical iron accumulation in early-onset (EOAD) compared to late-onset (LOAD) Alzheimer disease patients. However, the pattern and origin of iron accumulation is poorly understood. This study investigated the histopathological correlates of MRI contrast in both EOAD and LOAD. T2*-weighted MRI was performed on postmortem frontal cortex of controls, EOAD, and LOAD. Images were ordinally scored using predefined criteria followed by histology. Nonlinear histology-MRI registration was used to calculate pixel-wise spatial correlations based on the signal intensity. EOAD and LOAD were distinguishable based on 7T MRI from controls and from each other. Histology-MRI correlation analysis of the pixel intensities showed that the MRI contrast is best explained by increased iron accumulation and changes in cortical myelin, whereas amyloid and tau showed less spatial correspondence with T2*-weighted MRI. Neuropathologically, subtypes of Alzheimer's disease showed different patterns of iron accumulation and cortical myelin changes independent of amyloid and tau that may be detected by high-field susceptibility-based MRI.

Cortical Iron Reflects Severity of Alzheimer's Disease.

Abnormal iron distribution in the isocortex is increasingly recognized as an in vivo marker for Alzheimer's disease (AD). However, the contribution of iron accumulation to the AD pathology is still poorly understood. In this study, we investigated: 1) frontal cortical iron distribution in AD and normal aging and 2) the relation between iron distribution and degree of AD pathology. We used formalin fixed paraffin embedded frontal cortex from 10 AD patients, 10 elder, 10 middle aged, and 10 young controls and visualized iron with a modified Perl's histochemical procedure. AD and elderly subjects were not different with respect to age and sex distribution. Iron distribution in the frontal cortex was not affected by normal aging but was clearly different between AD and controls. AD showed accumulation of iron in plaques, activated microglia, and, in the most severe cases, in the mid-cortical layers along myelinated fibers. The degree of altered iron accumulations was correlated to the amount of amyloid-ß plaques and tau pathology in the same block, as well as to Braak stage (p < 0.001). AD and normal aging show different iron and myelin distribution in frontal cortex. These changes appear to occur after the development of the AD pathological hallmarks. These findings may help the interpretation of high resolution in vivo MRI and suggest the potential of using changes in iron-based MRI contrast to indirectly determine the degree of AD pathology in the frontal cortex.

Multiplex ligation-dependent probe amplification for the detection of 1p and 19q chromosomal loss in oligodendroglial tumors.

Multiplex ligation-dependent probe amplification (MLPA) is a new assay for the detection of multiple chromosomal deletions in tumor tissue in a single experiment. Since genotyping of gliomas with oligodendroglial features by the detection of 1p/19q chromosomal deletions became essential for treatment decisions, we developed and validated an MLPA-based assay to determine these losses in formalin fixed and paraffin embedded oligodendroglial tumors (OG). Nineteen OGs, and 10 control samples were analyzed by MLPA and the results were correlated with those obtained by fluorescent in situ hybridization (FISH). The MLPA results were reproducible in all samples in which repeated experiments were performed. In 18 of 19 OGs, MLPA and FISH were concordant for presence or absence of 1p deletion. In 3 OGs, MLPA detected a 19q deletion not shown by FISH. For the other 15 OGs, MLPA and FISH were concordant. In one sample with 50% to 75% of tumor, MLPA failed to detect the 1p/19q deletions revealed by FISH (though with borderline values of significance). We conclude that MLPA is a valid and reproducible method for the detection of 1p/19q chromosomal deletions in OGs stored on formalin fixed, paraffin embedded tissue.

Cervical deciduosis imitating dysplasia.

Ectopic cervical deciduosis is generally an accidental finding during pregnancy, and usually presents without any symptoms or need for therapeutic intervention. However, it can sometimes imitate dysplasia or carcinoma. We report a case of a 34-year-old G2P0, with a history of cervical dysplasia, presenting at 11 weeks of gestation, with vaginal blood loss. During examination, lesions mimicking dysplasia were found on the cervix. Histological examination reported cervical deciduosis. Deciduosis is a benign change during pregnancy and will resolve spontaneously. With the increasing use of cytology and colposcopy, the reported incidence is growing. When it is hard to differentiate between dysplasia and deciduosis, histological confirmation should be considered.

Comparison of histological techniques to visualize iron in paraffin-embedded brain tissue of patients with Alzheimer's disease.

Better knowledge of the distribution of iron in the brains of Alzheimer's disease (AD) patients may facilitate the development of an in vivo magnetic resonance (MR) marker for AD and may cast light on the role of this potentially toxic molecule in the pathogenesis of AD. Several histological iron staining techniques have been used in the past but they have not been systematically tested for sensitivity and specificity. This article compares three histochemical techniques and ferritin immunohistochemistry to visualize iron in paraffin-embedded human AD brain tissue. The specificity of the histochemical techniques was tested by staining sections after iron extraction. Iron was demonstrated in the white matter, in layers IV/V of the frontal neocortex, in iron containing plaques, and in microglia. In our hands, these structures were best visualized using the Meguro iron stain, a method that has not been described for iron staining in human brain or AD in particular. Ferritin immunohistochemistry stained microglia and iron containing plaques similar to the Meguro method but was less intense in myelin-associated iron. The Meguro method is most suitable for identifying iron-positive structures in paraffin-embedded human AD brain tissue.

Longitudinal monitoring of sex-related in vivo metabolic changes in the brain of Alzheimer's disease transgenic mouse using magnetic resonance spectroscopy.

Epidemiological studies indicate that the incidence of Alzheimer's disease (AD) is higher in women than in men. There is evidence that changes in metabolites in the brain associated with the development of AD are present earlier than structural brain changes. The effect of sex on the metabolic profile during the development of AD has not yet been studied. In this study we longitudinally monitored and compared in vivo metabolic changes in male and female AßPPswe, PSEN1dE9 transgenic mice brains using magnetic resonance spectroscopy. Our results show a lower level of glutamate as well as of N-acetylaspartate (NAA) in transgenic mice. The decline in NAA with age was more apparent in female mice. The level of taurine was higher in female mice and showed a faster decline over time. In conclusion, our study is the first to suggest that changes in the metabolic profile during AD development are influenced by sex.

MR microscopy of human amyloid-ß deposits: characterization of parenchymal amyloid, diffuse plaques, and vascular amyloid.

Cerebral deposits of amyloid-ß peptides (Aß) form the neuropathological hallmarks of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). In the brain, Aß can aggregate as insoluble fibrils present in amyloid plaques and vascular amyloid, or as diffuse plaques consisting of mainly non-fibrillar Aß. Previously, magnetic resonance imaging (MRI) has been shown to be capable of detecting individual amyloid plaques, not only via the associated iron, but also Aß itself has been suggested to be responsible for a decrease in the image intensity. In this current study we aim to investigate the MRI properties of the different cerebral Aß deposits including diffuse plaques and vascular amyloid. Postmortem 60-µm-thick brain sections of AD, CAA, and Down's syndrome patients, known to contain Aß, were studied. High resolution T2*- and T2-weighted MRI scans and quantitative relaxation maps were acquired using a microcoil on a Bruker 9.4T MRI system. Specific MRI characteristics of each type of Aß deposit were examined by co-registration of the MRI with Congo Red and Aß-immunostainings of the same sections. Our results show that only fibrillar Aß, present in both vascular and parenchymal amyloid, induced a significant change in T2* and T2 values. However, signal changes were not as consistent for all of the vessels affected by CAA, irrespective of possible dyshoric changes. In contrast, the non-fibrillar diffuse plaques did not create any detectable MRI signal changes. These findings are relevant for the interpretation and further development of (quantitative) MRI methods for the detection and follow-up of AD and CAA.

Glial reactions and the clearance of amyloid beta protein in the brains of patients with hereditary cerebral hemorrhage with amyloidosis-Dutch type.

Although the amyloid beta protein (Abeta) E693Q mutation enhances Abeta fibrillization in vitro and cerebral amyloid angiopathy (CAA) in vivo, brain parenchymal Abeta deposition and tau pathology in hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) are limited. To evaluate whether clearance of Abeta by glial cells may play a role in this regard, this immunohistochemical study of frontal cortex of 14 HCHWA-D autopsy brains was performed using double staining with glial markers and end-specific antibodies to Abetax-42 (Abeta42) and Abetax-40 (Abeta40). Tau pathology was also assessed. Numerous microglia and/or astrocytes carrying cytoplasmic Abeta42(+)40(-) granules were scattered among non-fibrillar (Congo red-negative) Abeta deposits, i.e., clouds, fine diffuse plaques, and Abeta42(+)40(-) dense diffuse plaques. On the other hand, activated microglia and reactive astrocytes associated with fibrillar (Congo red-positive) Abeta deposition, i.e., Abeta42(+)40(+) dense diffuse plaques and CAA invading the parenchyma, were virtually devoid of Abeta granules. Tau pathology was scant and most frequently associated with CAA. These results suggest that relatively non-fibrillar parenchymal Abeta deposits may be liable to glial clearance. Abeta sequestration by glial cells may be a factor limiting the levels of neurotoxic soluble Abeta oligomers in HCHWA-D brain.