Tissue microarray (TMA) use in post mortem neuropathology.

BACKGROUND: Tissue microarrays (TMAs), where each block (and thus section) contains multiple tissue cores from multiple blocks potentially allow more efficient use of tissue, reagents and time in neuropathology. NEW METHOD: The relationship between data from TMA cores and whole sections was investigated using 'virtual' TMA cores. This involved quantitative assessments of microglial pathology in white matter lesions and motor neuron disease, alongside qualitative TDP-43 inclusion status in motor neuron disease cases. Following this, a protocol was developed for TMA construction. RESULTS: For microglial pathology we found good concordance between virtual cores and whole sections for volume density using one 1.75 mm core (equivalent to a 2 mm core after accounting for peripheral tissue loss). More sophisticated microglial cell size and measures required two cores. Qualitative results of pTDP-43 pathology showed use of one 1.75 mm core gave a 100 % sensitivity and specificity within grey matter, and 88.3 % sensitivity and 100 % specificity within white matter. A method of producing the TMAs was suitable for immunohistochemistry both manually and by autostainer, with the minimal core loss from the microscope slide. COMPARISON WITH EXISTING METHODS: TMAs have been used infrequently in post mortem neuropathology research. However, we believe TMAs give comparable tissue assessment results and can be constructed, sectioned and stained with relative ease. CONCLUSIONS: We found TMAs could be used to assess both quantitative (microglial pathology) and qualitative pathology (TDP-43 proteinopathy) with greatly reduced quantities of tissue, time and reagents. These could be used for further work to improve data acquisition efficiency.

Review: Microglia in motor neuron disease.

Motor Neuron Disease (MND) is a fatal neurodegenerative condition, which is characterized by the selective loss of the upper and lower motor neurons. At the sites of motor neuron injury, accumulation of activated microglia, the primary immune cells of the central nervous system, is commonly observed in both human post mortem studies and animal models of MND. Microglial activation has been found to correlate with many clinical features and importantly, the speed of disease progression in humans. Both anti-inflammatory and pro-inflammatory microglial responses have been shown to influence disease progression in humans and models of MND. As such, microglia could both contribute to and protect against inflammatory mechanisms of pathogenesis in MND. While murine models have characterized the microglial response to MND, these studies have painted a complex and often contradictory picture, indicating a need for further characterization in humans. This review examines the potential role microglia play in MND in human and animal studies. Both the pro-inflammatory and anti-inflammatory responses will be addressed, throughout the course of disease, followed by the potential of microglia as a target in the development of disease-modifying treatments for MND.

TDP43 proteinopathy is associated with aberrant DNA methylation in human amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neurone (MN) degeneration and death. ALS can be sporadic (sALS) or familial, with a number of associated gene mutations, including C9orf72 (C9ALS). DNA methylation is an epigenetic mechanism whereby a methyl group is attached to a cytosine (5mC), resulting in gene expression repression. 5mC can be further oxidized to 5-hydroxymethylcytosine (5hmC). DNA methylation has been studied in other neurodegenerative diseases, but little work has been conducted in ALS. AIMS: To assess differences in DNA methylation in individuals with ALS and the relationship between DNA methylation and TDP43 pathology. METHODS: Post mortem tissue from controls, sALS cases and C9ALS cases were assessed by immunohistochemistry for 5mC and 5hmC in spinal cord, motor cortex and prefrontal cortex. LMNs were extracted from a subset of cases using laser capture microdissection. DNA from these underwent analysis using the MethylationEPIC array to determine which molecular processes were most affected. RESULTS: There were higher levels of 5mC and 5hmC in sALS and C9ALS in the residual lower motor neurones (LMNs) of the spinal cord. Importantly, in LMNs with TDP43 pathology there was less nuclear 5mC and 5hmC compared to the majority of residual LMNs that lacked TDP43 pathology. Enrichment analysis of the array data suggested RNA metabolism was particularly affected. CONCLUSIONS: DNA methylation is a contributory factor in ALS LMN pathology. This is not so for glia or neocortical neurones.

Neuropathological characterization of a novel TANK binding kinase (TBK1) gene loss of function mutation associated with amyotrophic lateral sclerosis.

AIMS: Mutations in TANK binding kinase gene (TBK1) are causative in amyotrophic lateral sclerosis (ALS), however correlations between clinical features and TBK1 mutations have not been fully elucidated. We aimed to identify and compare TBK1 mutations to clinical features in a cohort of ALS patients from Northern England. METHODS: TBK1 mutations were analysed in 290 ALS cases. Immunohistochemistry was performed in brain and spinal cord of one case with a novel in-frame deletion. RESULTS: Seven TBK1 variants were identified, including one novel in-frame deletion (p.85delIle). In silico analysis and literature suggested four variants were pathogenic, and three were variants of uncertain significance or benign. Post-mortem immunohistochemistry established an individual with the novel in-frame deletion had classical ALS and Type B FTLD-TDP pathology, with no changes in TBK1 staining or interferon regulatory factor IRF3. CONCLUSIONS: TBK1 mutations were present in 1.38% of our cohort, and screening showed no clear genotype-phenotype associations compared to other genetic and sporadic ALS cases. TBK1 immunohistochemistry was consistent with previously published literature and we are the first to show no differential expression of interferon regulatory factor IRF3, a downstream effector of TBK1 in the immune pathway, in the TBK1-mutant tissue, compared to controls.

Combined fused in sarcoma-positive (FUS+) basophilic inclusion body disease and atypical tauopathy presenting with an amyotrophic lateral sclerosis/motor neurone disease (ALS/MND)-plus phenotype.

AIMS: Amyotrophic lateral sclerosis/motor neurone disease (ALS/MND) is characterized by the presence of inclusions containing TDP-43 within motor neurones. In rare cases, ALS/MND may be associated with inclusions containing other proteins, such as fused in sarcoma (FUS), while motor system pathology may rarely be a feature of other neurodegenerative disorders. We here have investigated the association of FUS and tau pathology. METHODS: We report a case with an ALS/MND-plus clinical syndrome which pathologically demonstrated both FUS pathology and an atypical tauopathy. RESULTS: Clinical motor involvement was predominantly present in the upper motor neurone, and was accompanied by extrapyramidal features and sensory involvement, but with only minimal cognitive impairment. The presentation was sporadic and gene mutation screening was negative. Post mortem study demonstrated inclusions positive for FUS, including basophilic inclusion bodies. This was associated with 4R-tauopathy, largely as non-fibrillary diffuse phospho-tau in neurones, with granulovacuolar degeneration in a more restricted distribution. Double-staining revealed that neurones contained both types of protein pathology. CONCLUSION: FUS-positive basophilic inclusion body disease is a rare cause of ALS/MND, but in this case was associated with an unusual atypical tauopathy. The coexistence of two such rare neuropathologies raises the question of a pathogenic interaction.

Brain haemosiderin in older people: pathological evidence for an ischaemic origin of magnetic resonance imaging (MRI) microbleeds.

INTRODUCTION: Magnetic resonance imaging (MRI) cerebral microbleeds (CMB) arise from ferromagnetic haemosiderin iron assumed to derive from extravasation of erythrocytes. Light microscopy of ageing brain frequently reveals foci of haemosiderin from single crystalloids to larger, predominantly perivascular, aggregates. The pathological and radiological relationship between these findings is not resolved. METHODS: Haemosiderin deposition and vascular pathology in the putamen were quantified in 200 brains donated to the population-representative Medical Research Council Cognitive Function and Ageing Study. Molecular markers of gliosis and tissue integrity were assessed by immunohistochemistry in brains with highest (n = 20) and lowest (n = 20) levels of putamen haemosiderin. The association between haemosiderin counts and degenerative and vascular brain pathology, clinical data, and the haemochromatosis (HFE) gene H63D genotype were analysed. The frequency of MRI CMB in 10 cases with highest and lowest burden of putamen haemosiderin, was compared using post mortem 3T MRI. RESULTS: Greater putamen haemosiderin was significantly associated with putaminal indices of small vessel ischaemia (microinfarcts, P < 0.05; arteriolosclerosis, P < 0.05; perivascular attenuation, P < 0.001) and with lacunes in any brain region (P < 0.023) but not large vessel disease, or whole brain measures of neurodegenerative pathology. Higher levels of putamen haemosiderin correlated with more CMB (P < 0.003). CONCLUSIONS: The MRI-CMB concept should take account of brain iron homeostasis, and small vessel ischaemic change in later life, rather than only as a marker for minor episodes of cerebrovascular extravasation. These data are of clinical relevance, suggesting that basal ganglia MRI microbleeds may be a surrogate for ischaemic small vessel disease rather than exclusively a haemorrhagic diathesis.

Lack of unique neuropathology in amyotrophic lateral sclerosis associated with p.K54E angiogenin (ANG) mutation.

AIMS: Five to 10% of cases of amyotrophic lateral sclerosis are familial, with the most common genetic causes being mutations in the C9ORF72, SOD1, TARDBP and FUS genes. Mutations in the angiogenin gene, ANG, have been identified in both familial and sporadic patients in several populations within Europe and North America. The aim of this study was to establish the incidence of ANG mutations in a large cohort of 517 patients from Northern England and establish the neuropathology associated with these cases. METHODS: The single exon ANG gene was amplified, sequenced and analysed for mutations. Pathological examination of brain, spinal cord and skeletal muscle included conventional histology and immunohistochemistry. RESULTS: Mutation screening identified a single sporadic amyotrophic lateral sclerosis case with a p.K54E mutation, which is absent from 278 neurologically normal control samples. The clinical presentation was of limb onset amyotrophic lateral sclerosis, with rapid disease progression and no evidence of cognitive impairment. Neuropathological examination established the presence of characteristic ubiquitinated and TDP-43-positive neuronal and glial inclusions, but no abnormality in the distribution of angiogenin protein. DISCUSSION: There is only one previous report describing the neuropathology in a single case with a p.K17I ANG mutation which highlighted the presence of eosinophilic neuronal intranuclear inclusions in the hippocampus. The absence of this feature in the present case indicates that patients with ANG mutations do not always have pathological changes distinguishable from those of sporadic amyotrophic lateral sclerosis.

Size of hippocampal pyramidal neurons in schizophrenia.

BACKGROUND: Meta-analyses of hippocampal size have indicated that this structure is smaller in schizophrenia. This could reflect a reduction in the size of constituent neurons or a reduced number of neurons. AIMS: To measure the size of hippocampal pyramidal neurons in the brains of people with and without schizophrenia. METHOD: Pyramidal neuron size in hippocampal subfields was estimated stereologically from sections taken at 5 mm intervals throughout the whole length of right and left hippocampi from the brains of 13 people with schizophrenia and 16 controls. Results were assessed using repeated-measures analysis of covariance looking for a main effect of diagnosis and gender, and interactions of these with side. RESULTS: We were unable to detect significant differences related to diagnosis, gender or side for any hippocampal subfield for this series of cases. CONCLUSIONS: For this series of brains, hippocampal cell size is unchanged in schizophrenia.

Schizophrenia and the frontal lobes: post-mortem stereological study of tissue volume.

BACKGROUND: It has been suggested that there is frontal lobe involvement in schizophrenia, and that it may be lateralised and gender-specific. AIMS: To clarify the structure of the frontal lobes in schizophrenia in a post-mortem series. METHOD: The volume of white matter and cortical components of the frontal lobes was measured in brains of controls and patients with schizophrenia using planimetry and the Cavalieri principle. The components measured were: superior frontal gyrus, middle frontal gyrus, a composite of inferior frontal gyrus and orbito-frontal cortex, as well as total frontal lobe cortex and white matter. In addition, the anterior cingulate gyrus was measured. RESULTS: No diagnosis, gender, diagnosis x side, diagnosis x gender or diagnosis x gender x side interactions were observed in the volume of any of the components, the grey matter as a whole or the white matter. No evidence for volumetric inter-group differences was found for the anterior cingulate gyrus. CONCLUSIONS: Such structural abnormalities as are present in the frontal lobes are more subtle than straightforward alterations in tissue volume; they may include changes in shape and the pattern of gyral folding.

Temporal lobe epilepsy with and without psychosis: exploration of hippocampal pathology including that in subpopulations of neurons defined by their content of immunoreactive calcium-binding proteins.

We have investigated relationships between hippocampal/temporal lobe neuropathology and psychosis in subjects with temporal lobe epilepsy, paying particular attention to possible differences in density of hippocampal neurons immunoreactive for calcium-binding proteins. There was a trend for a greater prevalence of left handedness in the psychotic (n = 6) than the non-psychotic (n = 26) cases (P = 0.0504). Psychotic cases also differed from non-psychotic ones in having: (1) more focal lesions outside the hippocampus (P = 0.006); (2) less severe CAI neuron loss (P = 0.015); and (3) a trend, after Bonferroni correction, for a higher density of calbindin-immunoreactive neurons in the CA4 (P = 0.022). An additional finding was that dentate granule cell dispersion was significantly associated with the presence of a reduced density of calretinin-immunoreactive neurons in CA4 (P = 0.002) and with a more severe loss of CA4 neurons visible with Nissl stain (P = 0.003). Thus, cases of temporal lobe epilepsy with psychosis were distinguishable on the basis of a higher density of calbindin-reactive neurons in CA4 as well as on more general aspects of their pathology.

Anomalous asymmetry of fusiform and parahippocampal gyrus gray matter in schizophrenia: A postmortem study.

OBJECTIVE: Anomalies of structure and asymmetry of the parahippocampal gyrus (origin of the perforant path input to the hippocampal formation in the medial temporal lobe) have been shown in some postmortem studies of schizophrenia, but previous studies have not included the fusiform gyrus (which may have a role in facial recognition and naming), adjacent to the parahippocampal gyrus on the ventral occipitotemporal surface. METHOD: The volumes of gray matter in the left and right parahippocampal and fusiform gyri were assessed with a stereological point-counting technique in the temporal lobes from formalin-fixed brains of 27 comparison subjects and 31 patients with schizophrenia. Age was a covariate and gender was a factor in the analysis. RESULTS: In relation to the comparison subjects, the schizophrenic patients (both sexes) had lower volumes of both the parahippocampal and fusiform gyri on the left side. For both structures a left-greater-than-right volume asymmetry was present in the comparison subjects, but this asymmetry was reversed in the parahippocampal and fusiform gyri of the schizophrenic patients. A sex difference was present with respect to age at onset-degree of anomaly of asymmetry for both gyri increased with age at onset in men but not in women. CONCLUSIONS: The findings add substance to the view that the sex-related dimension of symmetry/asymmetry is integral to the disease process in schizophrenia and draw attention to the fusiform gyrus as a structure of particular interest in relation to disturbances of identification and naming in psychosis.

Fiber content of the fornix in schizophrenia: lack of evidence for a primary limbic encephalopathy.

OBJECTIVE: There have been claims that schizophrenia is a disease of the limbic circuit and that the volume of the hippocampus and its content of neurons are low in schizophrenia. The fornix is a major pathway through which neurons project to and from the hippocampus. The authors investigated whether the fiber number or structure of the fornix is abnormal in schizophrenia, as was suggested by an earlier MRI study. METHOD: A section of fornix was removed from each hemisphere of postmortem brains of 16 male and 13 female schizophrenic patients and a comparison group of 19 men and 14 women. Cross-sectional area, fiber density, and total fiber number were examined for differences between diagnostic groups and between genders. RESULTS: The men had a lower fiber density in the fornix than the women. Fiber density on the left side was greater in the schizophrenic men than in the comparison men. For total fiber number (density multiplied by area) there were no differences between groups. Density was found to decrease with increasing area, suggesting that these measures may be affected by degree of myelination. CONCLUSIONS: The fornix does not show the abnormalities in cross-sectional area or total fiber number that would be expected if the primary impact of schizophrenia is on the hippocampus and limbic system. The greater density on the left in schizophrenic men suggests an effect of schizophrenia on myelination related to sex and asymmetry, which may reflect one aspect of a global delay in brain development.

The size and fiber composition of the anterior commissure with respect to gender and schizophrenia.

BACKGROUND: In light of evidence for deviations in asymmetry and alterations in the anatomy of the corpus callosum in schizophrenia, this study examined the anterior commissure in post mortem brains (n = 14 female control patients, 15 male control patients, 11 female schizophrenic patients, 15 male schizophrenic patients). METHODS: Measures were made of the cross-sectional area of the anterior commissure in the midsagittal plane. In addition, the fiber density and fiber number were measured in a subset of cases (n = 10 female control subjects, 10 male control subjects, 8 female schizophrenic patients, 9 male schizophrenic patients), using the Palmgren silver stain and stereological methods. RESULTS: In control subjects, fiber numbers were greater (p = .024) in women than men. In schizophrenia, the cross-sectional area was unaffected, but for fiber density there was a significant gender x diagnosis interaction (p = .026), corresponding to a reduction in female, but not male patients with schizophrenia. CONCLUSIONS: The reduction in density of fibers in the anterior commissure is consistent with an alteration of interhemispheric connectivity in schizophrenia, but the restriction of the finding to women emphasizes the relevance of gender to understanding the nature of the hemispheric interaction.

The size and fibre composition of the corpus callosum with respect to gender and schizophrenia: a post-mortem study.

In this study the cross-sectional area (in n = 14 female controls, 15 male controls, 11 female patients with schizophrenia, 15 male patients with schizophrenia) and fibre composition (in n = 11 female controls, 10 male controls, 10 female patients with schizophrenia, 10 male patients with schizophrenia) of the corpus callosum in post-mortem control and schizophrenic brains was examined. A gender x diagnosis interaction (P = 0.005) was seen in the density of axons in all regions of the corpus callosum except the posterior midbody and splenium. Amongst controls, females had greater density than males; in patients with schizophrenia this difference was reversed. A reduction in the total number of fibres in all regions of the corpus callosum except the rostrum was observed in female schizophrenic patients (P = 0.006; when controlling for brain weight, P = 0.053). A trend towards a reduced cross-sectional area of the corpus callosum was seen in schizophrenia (P = 0.098); however, this is likely to be no more than a reflection of an overall reduction in brain size. With age, all subregions of the corpus callosum except the rostrum showed a significant reduction in cross-sectional area (P = 0.018) and total fibre number (P = 0.002). These findings suggest that in schizophrenia there is a subtle and gender-dependent alteration in the forebrain commissures that may relate to the deviations in asymmetry seen in other studies, but the precise anatomical explanation remains obscure.

Schizophrenia and temporal lobe asymmetry. A post-mortem stereological study of tissue volume.

BACKGROUND: A previous report by Crow of a left-sided increase in temporal horn volume in schizophrenia implies a left-sided loss of tissue. AIMS: To elucidate the structural nature of schizophrenia. METHOD: The volume of grey matter in the temporal pole and inferior, middle and superior temporal gyri was measured, in addition to the total volume of grey and white matter, in the temporal lobes of the brains of 29 patients with schizophrenia and 27 controls. RESULTS: We found a significant left-sided reduction in the superior temporal gyrus in both males and females with schizophrenia, which was related to increasing age of onset in the males. The total volume of temporal lobe grey and white matter was also significantly reduced. Although being more marked on the left than the right, the lateralisation for these total grey and white measures (by contrast with the superior temporal gyrus alone) did not attain formal statistical significance. CONCLUSIONS: Confirmation of a lateralised reduction in the superior temporal gyrus, which is differentially related to age of onset according to gender, adds to evidence that the changes in schizophrenia are in systems that are lateralised. The findings implicate language as the relevant function.

Temporal-lobe length is reduced, and gyral folding is increased in schizophrenia: a post-mortem study.

This post-mortem study of the brains of 29 controls and 25 patients with schizophrenia investigated the length and gyral folding of the temporal lobes, and the asymmetries and inter-relationships of these two measures. The degree of gyral folding was significantly increased in schizophrenia (p = 0.002), but the orientation of the sulci was not changed (p = 0.420). Neither gender nor side affected any of the measures of gyral anatomy, nor were there any significant interactions of these variables with diagnosis. The temporal lobes were significantly shortened in schizophrenia, on two different measures (p = 0.009, and p = 0.001), and on one of these, females had shorter temporal lobes than males (p < 0.0005). No diagnosis x side interactions were found. The temporal-lobe shortening remained after controlling for brain weight and was not statistically related to gyral folding. These two structural changes may reflect an alteration of the cortico-cortical connectivity of the brain in schizophrenia.

Anomalies of cerebral asymmetry in schizophrenia interact with gender and age of onset: a post-mortem study.

In a post-mortem study of cerebral asymmetry in schizophrenia it was found that asymmetry of the length from the frontal pole to the central sulcus measured dorsally over the external surface of the brain on both hemispheres, showed a gender x diagnosis interaction (p = 0.002). Female controls had a left-greater-than-right asymmetry, and the male controls had a right-greater-than-left asymmetry. This pattern was reversed in schizophrenia. The converse effect was observed on a similar measure of the occipito-parietal lobes (p = 0.028). Significant changes were not seen in measures taken around the lateral surface of the hemispheres. Further, within the patient group, the frontal lobe asymmetry was related to age of onset such that leftward asymmetrical brains were associated with a later age of onset than rightward asymmetrical brains (p = 0.0463 for the females; p = 0.0162 for the males). The occipito-parietal asymmetry was not related to age of onset. We conclude that the asymmetry of the relative distribution of tissue between frontal and posterior regions of the hemispheres is altered in schizophrenia. The findings also suggest that there is an interaction between gender and cerebral asymmetry that is critical in determining age of onset.