Human brain tissue microarrays as a platform to investigate diseases of the nervous system

We constructed tissue microarray (TMA) blocks containing post-mortem human brain tissue from subjects with clinically and neuropathologically verified Alzheimer's disease (AD), corticobasal degeneration (CBD), progressive supranuclear palsy, Lewy body disease, multisystem atrophy (MSA) as well as an age matched control. Fifteen donor blocks were merged into two TMA blocks containing 72, 2-mm punch core samples with representative brain regions generally affected in degenerative disorders. Hyperphosphorylated-gamma, alpha-synuclein and beta-amyloid-related pathologies were estimated. The diseases were easily recognized by evaluating the two TMA sections and the results assessing TMA sections were comparable with the assessment of the whole brain sections. The assessment of TMA sections revealed concomitant multifocal alpha-synuclein pathology in AD, mild tau-involvement in the case of MSA and a slight AD-type pathology in the case of CBD. These findings emphasize the importance of searching for a variety of pathologies in "the whole brain" rather than restricting the examination to a few vulnerable regions. Furthermore, the TMA methodology clearly reduced the number of sections needed for evaluating the whole brain, it increased the amount of research material generated and furthermore no detailed neuroanatomical knowledge was required for assessment of data.     

Decreasing myelin density reflected increasing white matter pathology in Alzheimer's disease--a neuropathological study

BACKGROUND: White matter disease (WMD) is frequently seen in Alzheimer's disease (AD) at neuropathological examination. It is defined as a subtotal tissue loss with a reduction of myelin, axons and oligodendrocytes as well as astrocytosis. Studies quantitatively defining the myelin loss in AD are scarce. The aim was to develop a method that could provide numerical values of myelin density in AD. The purpose was to compare the myelin contents in increasing grades of pathology of WMD, with age and cortical AD pathology as well as in different regions of the brain in AD. MATERIAL AND METHODS: Sixteen cases with AD and concomitant WMD were investigated with an in-house developed image analysis technique to determine the myelin attenuation with optical density (OD) in frontoparietal, parietal, temporal and occipital white matter on whole brain coronal sections stained for myelin with Luxol Fast Blue (LFB). The OD values in LFB were compared grouped according to Haematoxylin/Eosin (HE) evaluated mild, moderate and severe WMD or normal tissue. The OD values were also correlated with age and cortical AD pathology and compared between the different studied white matter regions. RESULTS: Increasing severity of WMD was associated with a statistically significant OD reduction. No correlation was seen between age and OD or overall cortical AD pathology. The OD values were significantly lower in frontoparietal-compared to occipital white matter. CONCLUSIONS: Myelin loss in AD with WMD is a marked morphologic component of the disease and it is possible to determine the reduction objectively in neuropathological specimens with quantitative measures. This may be of use for clinical diagnostics including brain imaging.     

Oxidative DNA damage in the spinal cord in multiple sclerosis

Alzheimer's disease (AD) and vascular dementia (VaD) are two of the common dementias in the elderly. Imaging studies have demonstrated white matter changes of vascular etiology to occur in both disorders. While there have increased efforts to differentiate white matter pathology by quantitative magentic resonance imaging in these disorders there is no clear consensus. We assessed white matter lesions in brains of the first 100 demented patients who came to autopsy from our prospectively evaluated series. We examined the extent of axonal pathology by conventional methods including LFB for myelin, and amyloid-β precursor protein (AβPP) immunocytochemistry with antibodies to 22C11. AβPP has recently been used to examine axonal injury in head trauma. Our results indicated AβPP reactive accumulation along white matter tracts in the temporal cortex encompassing the hippocampal formation to be common. The highest reactivity was evident in tissue from VaD subjects compared with that from AD or dementia with Lewy bodies (DLB). Compared with conventional myelin stains, AβPP immunocytochemistry was more sensitive to visualize subtle changes. We suggest AβPP immunocytochemistry is a useful index to assess ischaemic white matter lesions to differentiate dementias.
Acknowledgements:  Supported by the MRC (UK), Alzheimer's Research Trust (UK) and Alzheimer's Association (USA).     

Astroglial localization of cholesteryl ester transfer protein in normal and Alzheimer's disease brain tissues

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that facilitates the transfer of cholesteryl esters, phospholipids and triglycerides between the lipoproteins, and regulates plasma high-density lipoprotein levels. We examined CETP-like immunolabeling in non-neurological and Alzheimer's disease (AD) liver and brain tissues. The anti-CETP antibodies showed positive staining in round cells in the liver sinusoid and in brain astrocytes. In the brains of non-neurological cases, positively stained astrocytes were preferentially distributed in the white matter. In AD tissue, many reactive astrocytes in the gray matter as well as the white matter astrocytes had CETP-like immunoreactivity. CETP-positive astrocytes may play a role for AD pathology such as tissue repair.     

In vivo demonstration of microstructural brain pathology in progressive supranuclear palsy: A DTI study using TBSS

We investigated DTI changes, potentially indicating alterations of microstructure and brain tissue integrity in 13 patients with probable progressive supranuclear palsy (PSP, Richardson syndrome) at stage III or less and 10 age‐matched controls using a whole brain analysis of diffusion tensor imaging (DTI) data. DTI images were analyzed using tract‐based spatial statistics, a hypothesis‐free technique. Fractional anisotropy (FA), radial diffusivity (RD), and axial diffusivity (AD) were determined. In patients with PSP, significant increases in FA (P < 0.0001), an unspecific measure of microstructural tissue integrity, were found in the cerebellum and in the superior cerebellar peduncle bilaterally, in the fornix, the body of the corpus callosum and the olfactory region, when compared with age‐matched healthy controls. Further, regional reductions in AD (P < 0.0001), an indicator of altered axonal integrity, were observed in the pons, the right substantia nigra and the cerebellar white matter bilaterally. Significant increases in RD (P < 0.0001), a potential measure of altered myelin integrity, occurred bilaterally in the superior cerebellar peduncle, the cerebellar white matter, the vermis of the cerebellum, the fornix, the body of the corpus callosum, and the olfactory region. RD values in the superior cerebellar peduncle discriminated patients with PSP and controls with high sensitivity (0.92) and specificity (1.0). The findings are supported by neuropathological studies. Our data suggest the usefulness of this clinically available new technique as a possible tool for differential diagnosis. © 2010 Movement Disorder Society     

Proton magnetic resonance spectroscopy in brain white matter disorders

The advent of magnetic resonance imaging (MRI) has revolutionized the clinical approach to the evaluation of brain white matter disorders and has contributed significantly to expansion of the concept of these diseases. MRI is very sensitive at detecting white matter lesions, but conventional T1 and T2-weighted images do not provide specific pathological information about the lesions, and correlation between MRI lesion load and clinical disability is often weak. Proton magnetic resonance spectroscopy can provide chemical-pathological information of a given tissue invivo. The use of this MR technique in brain white matter disorders has shown to improve diagnostic classification and to provide surrogate measures useful for monitoring disease evolution and response to therapeutic intervention.The study was supported in part by grants from the Multiple Sclerosis Society of Canada and from the Progetto Sclerosi Multipla of the Istituto Superiore di Sanità, Rome, Italy.     

Distinct white matter injury associated with medial temporal lobe atrophy in Alzheimer's versus semantic dementia

This study aims at further understanding the distinct vulnerability of brain networks in Alzheimer's disease (AD) versus semantic dementia (SD) investigating the white matter injury associated with medial temporal lobe (MTL) atrophy in both conditions. Twenty‐six AD patients, twenty‐one SD patients, and thirty‐nine controls underwent a high‐resolution T1‐MRI scan allowing to obtain maps of grey matter volume and white matter density. A statistical conjunction approach was used to identify MTL regions showing grey matter atrophy in both patient groups. The relationship between this common grey matter atrophy and white matter density maps was then assessed within each patient group. Patterns of grey matter atrophy were distinct in AD and SD but included a common region in the MTL, encompassing the hippocampus and amygdala. This common atrophy was associated with alterations in different white matter areas in AD versus SD, mainly including the cingulum and corpus callosum in AD, while restricted to the temporal lobe — essentially the uncinate and inferior longitudinal fasciculi — in SD. Complementary analyses revealed that these relationships remained significant when controlling for global atrophy or disease severity. Overall, this study provides the first evidence that atrophy of the same MTL region is related to damage in distinct white matter fibers in AD and SD. These different patterns emphasize the vulnerability of distinct brain networks related to the MTL in these two disorders, which might underlie the discrepancy in their symptoms. These results further suggest differences between AD and SD in the neuropathological processes occurring in the MTL. Hum Brain Mapp 38:1791–1800, 2017. © 2017 Wiley Periodicals, Inc.     

Structural brain abnormalities in unselected in-patients with major depression

OBJECTIVE: Several studies have indicated an increased frequency of cerebral atrophy and white matter lesions in patients with major depression, especially in older age groups. METHOD: Forty-four representative in-patients with major depression in which neurological disorders were clinically excluded, and 49 age- and gender-matched controls were MR scanned. RESULTS: Unexpectedly, two of the patients had severe brain pathology which could account for their psychiatric symptoms. Analysis of the remaining patients (mean age 42 years) did not reveal an increased frequency of cerebral atrophy. The number of white matter lesions increased with age to an odds ratio greater than 3 for patients aged 50, but this was not statistically significant. CONCLUSION: Brain atrophy and white matter lesions did not occur with significantly increased frequency in these relatively young unselected depressives, but the finding of severe brain pathology stresses the importance of brain imaging in late-onset psychiatric disorders.     

Neuropathological evaluation of mixed dementia

Mixed dementia (MD) refers to a combination of definite Alzheimer disease (AD) and vascular encephalopathy, but the distinction between both disorders is controversial. For the diagnosis of MD the clinical/neuroimaging criteria of possible AD plus cerebrovascular disease (CVD) as separate entities are used, but causal relations between vascular brain lesions and dementia are unclear. We proposed the combination of autopsy-proven AD with multiple vascular or ischemic lesions with about 30-50 ml of infarcted/damaged brain tissue. The population-based prevalence of MD is unknown. In retrospective and prospective autopsy studies, it ranges from 2 to 58% with reasonable means of 6-12%. In a consecutive autopsy series of 1500 demented elderly subjects, 830 of which with clinically probable AD, in Vienna, Austria, 41.5 to 52.0% showed "pure" AD, 7% atypical AD, 16-20% AD plus cerebrovascular lesions, and 9% AD plus Lewy body pathology; MD was diagnosed in 4.6 and 2.4%, and "pure" vascular dementia (VaD) in 11 and 2.0%, respectively, while 16.3/6.1% were other dementing disorders, and 1% showed no specific pathology. Like the MRC-CFAS and other studies, this indicates frequent coexistence of AD with multiple cerebrovascular lesions in cognitively impaired patients. In both AD and VaD, vascular lesions frequently involved subcortical regions (basal ganglia, thalamus, hippocampus, and white matter) or were multiple microinfarcts, whereas in MD, large/hemispheral infarcts and multiple microinfarcts were more frequent, suggesting different pathogenic mechanisms. In early/mild AD, critically located small vascular lesions may induce/promote cognitive decline, but in full-blown AD they appear of minor importance. Discussion of the major pathogenic factors inducing AD, VaD and MD suggests synergistic relations between these disorders. However, currently available morphological criteria for AD and VaD are of limited value for the diagnosis of MD and generally accepted and validated histopathological criteria for the diagnosis of VaD and MD are currently not available. Therefore, more distinct and critically evaluated clinico-pathological criteria are warranted.     

Diffusion tensor imaging in early Alzheimer's disease

Our aim was to investigate the extent of white matter tissue damage in patients with early Alzheimer disease (AD) using diffusion tensor magnetic resonance imaging (DTI). Although AD pathology mainly affects cortical grey matter, previous magnetic resonance imaging (MRI) studies showed that changes also exist in the white matter (WM). However, the nature of AD-associated WM damage is still unclear. Conventional and DTI examinations (b=1000 s/mm(2), 25 directions) were obtained from 12 patients with early AD (Mini Mental State Examination [MMSE] score=27, Grober and Buschke test score=33.2, digit span score=5.6) and 12 sex- and age-matched volunteers. The right and left mean diffusivity (MD) and fractional anisotropy (FA) of several WM regions were pooled in each patient and control, and compared between the two groups. Volumes of the whole brain and degree of atrophy of the temporal lobe were compared between the two groups. In AD, MD was increased in the splenium of the corpus callosum and in the WM in the frontal and parietal lobes. FA was bilaterally decreased in the WM of the temporal lobe, the frontal lobe and the splenium compared with corresponding regions in controls. Values in other areas (occipital area, superior temporal area, cingulum, internal capsule, and genu of the corpus callosum) were not different between patients and controls. No correlations were found between the MMSE score and the anisotropy indices. Findings of DTI reveal abnormalities in the frontal and temporal WM in early AD patients. These changes are compatible with early temporal-to-frontal disconnections.     

Observation of serum proteins in the edematous brain tissue by fluorescein-antibody technique

Summary Histologic distribution of serum proteins within normal and edematous brain tissues was investigated with the fluorescein-antibody technique. Brain edema was produced in dogs with cold injury, as described byKlatzo et al. In normal brain large amounts of serum proteins were restricted to the vascular lumina and the presence of these proteins was sparse in nerve, glia and endothelial cells. On the other hand, edematous brain tissue contained large quantities of serum proteins; they were found in larger amounts in white than in grey matter, and were more abundant in white matter in immediate proximity to the lesion than in the area more distant from it. In the edematous white matter near the cold injury in cerebral cortex, serum proteins were distributed diffusely so that no relationship to the histological structures was noted. In the periphery of the edematous white matter they were distributed in reticular fashion, which might indicate that they are present in the net work of cell processes. Accumulation of serum proteins was also noted in the Virchow-Robin space as well as around the small vessels.We wish to thank Dr.Y. Hamashima, Associate Professor of the Department of Pathology, Kyoto University Medical School for his invaluable assistance with the fluorescentantibody technique and discussion of the results.     

Voxel-based morphometry in Alzheimer's disease

Recent morphometric MRI studies have investigated brain volume abnormalities associated with the diagnosis of Alzheimer's disease (AD) using voxel-based morphometry (VBM). This technique allows the assessment of gray matter volumes in subjects with AD or related conditions compared with healthy controls in an automated fashion, across the whole brain. This article reviews VBM findings related to different AD stages and its prodrome, mild cognitive impairment. These findings include not only gray matter deficits in medial temporal structures as seen in former MRI studies of AD conducted using manual region-of-interest measurements, but also volume changes in several other brain regions not assessed in previous MRI studies. We also discuss potential applications of VBM to improve AD diagnostic accuracy in routine clinical practice. Finally, we highlight future research directions in this field, including: investigations on the relationship between VBM findings of multifocal gray matter deficits and changes in white matter tracts that interconnect such regions; the need for population-based VBM studies using large AD samples; and the potential of studies combining VBM measurements with other potential biological markers (such as brain imaging indices of amyloid-beta deposition and cerebrospinal fluid AD markers) to further advance our knowledge about the physiopathology of AD.     

Increase of white matter string vessels in Alzheimer's disease

String vessels are collagenous structures connected to capillaries. They have no endothelial cells or lumen. We assessed collagen IV-labeled string vessels in the white matter (WM) of subjects with Alzheimer's disease (AD) (n = 12) and non-AD controls (n = 11) using 100 microm celloidin sections. Ten standard fields were digitally captured and the number and length of normal vessels and string vessels were quantified by computerized image analysis. The WM of the AD-diagnosed individuals contained more strings per mm2 (3.95 +/- 0.49) than comparable WM from controls (1.36 +/- 0.39) (p = 0.0005) and had increased total string vessel length in mm/mm2 (AD = 0.29 +/- 0.04; control = 0.10 +/- 0.03; p = 0.0015). There was a 25% increase (not statistically significant) in vessel density in mm/mm2 in AD subjects (AD = 11.88 +/- 0.87; control = 9.53 +/- 0.78; p = 0.06), presumably due to brain atrophy in the white matter. Although vessel length was slightly increased in AD subjects, they still had more than double the string length per total vessel length (AD = 2.88 +/- 0.38) compared to controls (1.36 +/- 0.27) (p = 0.0057). This increase in string vessels in the white matter of AD subjects suggests a decrease in vascular supply in this disease.     

The pathology of "vascular dementia": a critical update

The prevalence, morphology and pathogenesis of vascular dementia (VaD), recently termed vascular cognitive disorder (VCD), are a matter of discussion.VaD is suggested in 8-15% of cognitively impaired aged subjects. Its prevalence in autopsy series ranges from 0.03 to 58% (mean 8-15% in Western series, 22-35% in Japan). Neuropathology shows multifocal and/or diffuse lesions, ranging from lacunes and microinfarcts, often involving subcortical and strategically important brain areas (thalamus, frontobasal, limbic system), white matter lesions and hippocampal sclerosis to multi-infarct encephalopathy and diffuse post-ischemic lesions. They result from systemic, cardiac and local large and small vessel disease. Pathogenesis is multifactorial and pathophysiology affects neuronal networks involved in cognition, behavior, execution and memory. Vascular lesions often coexist with Alzheimer's disease (AD) and other pathologies. Minor vascular lesions hardly contribute to cognitive decline in full-blown AD, while both mild Alzheimer pathology and small vessel disease interact synergistically. AD pathology is less severe in the presence of vascular lesions. The lesion pattern in "pure" VaD/VCD) related to microangiopathies differs from that in "mixed dementia" (AD + vascular encephalopathy), often associated with large infarcts, suggesting different pathogenesis. Due to the heterogeneity of cerebrovascular pathology and its causative factors, no validated neuropathologic criteria for VaD are currently available, and a large variability across laboratories still exists in morphologic examination procedures and techniques. Further prospective clinico-pathologic studies are needed to validate diagnostic criteria for VaD and to clarify the impact of vascular lesions on cognitive impairment.     

Contribution of Brain Imaging to the Understanding Of Gait Disorders in Alzheimer's Disease: A Systematic Review

Although gait disorders are common in Alzheimer's disease (AD), determining which brain structures and related lesions are specifically involved is a goal yet to be reached. Our objective was to systematically review all published data that examined associations between gait disorders and brain imaging in AD. Of 486 selected studies, 4 observational studies met the selection criteria. The number of participants ranged from 2 to 61 community dwellers (29%-100% female) with prodromal or dementia-stage AD. Quantitative gait disorders (ie, slower gait velocity explained by shorter stride length) were associated with white matter lesions, mainly in the medial frontal lobes and basal ganglia. The nigrostriatal dopamine system was unaffected. Qualitative gait disorders (ie, higher stride length variability) correlated with lower hippocampal volume and function. Gait disorders in AD could be explained by a high burden of age-related subcortical hyperintensities on the frontal-subcortical circuits (nonspecific) together with hippocampal atrophy and hypometabolism (specific).     

Regional and Cellular Localization of Presenilin-2 RNA in Rat and Human Brain

In situhybridization probes selective for presenilin-2 (PS-2) were used to determine the regional and cellular expression pattern of PS-2 mRNA in rat and human brain. In rat brain, the greatest expression of PS-2 mRNA is in the granule cell layers of the dentate gyrus and cerebellum. Molecular layers within these structures are virtually devoid of signal. Cortical expression of PS-2 message is restricted to neuronal layers, while the hybridization signal is weak or absent in molecular layers and white matter. Kidney, liver, and spleen display moderate levels of PS-2 message. A PS-2 sense strand probe produced no specific signals in any tissue. In human brain, the greatest hybridization signal for PS-2 is present in the granule cells of the cerebellum. Within hippocampus, the granule cell layer of dentate is strongly labeled, with CA3 pyramidal neurons also clearly visible. A laminar expression pattern is seen in the neuronal layers of human frontal and temporal cortex, with the deeper laminae having the strongest signals. These data are consistent with a primarily neuronal localization of PS-2 mRNA within the brains of both rat and human. Within the limitations of the analysis, it appears that virtually every neuron is labeled, and differences in the intensity of labeling are associated with both neuron size/density and brain region. The distribution of PS-2 RNA is not restricted to those regions having the greatest pathology in Alzheimer's disease. However, one unusual pathological feature of PS-2 mutations causing AD is the presence of cerebellar amyloid plaques in some cases. It is intriguing, in this context, that PS-2 RNA is enriched in the cerebellum, especially in human specimens.     

Ultrastructural hippocampal and white matter alterations in mild cognitive impairment: a diffusion tensor imaging study

Mild cognitive impairment (MCI) is considered to be a transitional stage between normal aging and dementia. In Alzheimer's disease (AD), white matter structural pathology is due to Wallerian degeneration and central angiopathy. However, in MCI patients, the presence and extent of white matter alterations as a possible correlate of impaired memory function and as predictor of subsequent progression to AD is not clarified yet. Diffusion tensor imaging (DTI) reveals the ultrastructural integrity of cerebral white matter tracts. Therefore, it could detect pathological processes that modify tissue integrity in patients with MCI. In our prospective study, conventional and diffusion tensor MR scans were obtained from 14 patients with MCI, 19 patients with AD, and 10 healthy controls. Mean diffusivity (MD) and fractional anisotropy (FA) were measured in temporal, frontal, parietal and occipital white matter regions as well as in the corpus callosum (genu and splenium) and the hippocampus. MCI patients showed higher MD values in the left centrum semiovale (p = 0.013; right: p = 0.026), in the left temporal (p = 0.006), the right temporal (p = 0.014) and the left hippocampal (p = 0.002) region as compared to the control group. FA values of MCI patients and controls did not differ significantly in any region. Compared to controls, AD patients had increased MD values in the left centrum semiovale (p = 0.012), the left parietal (p = 0.001), the right parietal (p = 0.028), the left temporal (p = 0.018), the right temporal (p = 0.011) and the left hippocampal region (p = 0.002). Decreased FA values were measured in the left temporal area (p = 0.017) and in the left hippocampus (p = 0.031) in AD patients compared to controls. FA and MD values did not differ significantly between AD and MCI patients. Elevated MD values indicating brain tissue alterations in MCI patients were found in regions that are typically involved in early changes due to AD, particularly the left hippocampus. The sensitivity of distinguishing MCI patients from controls was 71.4% (with a specificity set at 80%). Therefore, the DTI technique validates the MCI concept, and diffusion tensor MR measurement can be a helpful tool to quantify MCI pathology in vivo.     

Proton MR spectroscopy to assess axonal damage in multiple sclerosis and other white matter disorders

Proton MR spectroscopy allows in vivo measurement of N-acetylaspartate in white matter, providing a biochemical index of axonal integrity. Several recent studies of patients with multiple sclerosis and other white matter disorders have shown both transient and sustained decreases in N-acetylaspartate in white matter lesions and in brain regions appearing normal on conventional MRI. These data have emphasised that a substantial amount of axonal damage or loss (presumably secondary to myelin pathology) is consistently present in most of these disorders. Recent post-mortem studies support these results. In contrast to changes seen with conventional MR imaging, decreases in N-acetylaspartate have shown a close correlation with changes in neurological status. This suggests that axonal damage may be more relevant than demyelination for determining chronic functional impairments in primary white matter diseases. Thus, serial measurement of brain N-acetylaspartate with proton MR spectroscopy can provide a reliable and clinically-relevant monitor of disease evolution. As pathological changes responsible for long-term morbidity are logically important targets for therapeutic agents, early treatment directed at axonal protection should be useful in these disorders.     

Selective reduction of N-acetylaspartate in medial temporal and parietal lobes in AD

BACKGROUND: Both AD and normal aging cause brain atrophy, limiting the ability of MRI to distinguish between AD and age-related brain tissue loss. MRS imaging (MRSI) measures the neuronal marker N-acetylaspartate (NAA), which could help assess brain change in AD and aging. OBJECTIVES: To determine the effects of AD on concentrations of NAA, and choline- and creatine-containing compounds in different brain regions and to assess the extent NAA in combination with volume measurements by MRI improves discrimination between AD patients and cognitively normal subjects. METHODS: Fifty-six patients with AD (mean age: 75.6 +/- 8.0 years) and 54 cognitively normal subjects (mean age: 74.3 +/- 8.1 years) were studied using MRSI and MRI. RESULTS: NAA concentration was less in patients with AD compared with healthy subjects by 21% (p < 0.0001) in the medial temporal lobe and by 13% to 18% (p < 0.003) in parietal lobe gray matter (GM), but was not changed significantly in white matter and frontal lobe GM. In addition to lower NAA, AD patients had 29% smaller hippocampi and 11% less cortical GM than healthy subjects. Classification of AD and healthy subjects increased significantly from 89% accuracy using hippocampal volume alone to 95% accuracy using hippocampal volume and NAA together. CONCLUSION: In addition to brain atrophy, NAA reductions occur in regions that are predominantly impacted by AD pathology.