Extensive grey matter pathology in the cerebellum in multiple sclerosis is linked to inflammation in the subarachnoid space

Multiple Sclerosis (MS) is a progressive inflammatory neurological disease affecting myelin, neurons and glia. Demyelination and neurodegeneration of cortical grey matter contributes to a more severe disease and inflammation of the forebrain meninges associates with pathology of the underlying neocortical grey matter, particularly in deep sulci. We assessed the extent of meningeal inflammation of the cerebellum, another structure with a deeply folded anatomy, to better understand the association between subarachnoid inflammation and grey matter pathology in progressive MS.We examined demyelinating and neuronal pathology in the context of meningeal inflammation in cerebellar tissue blocks from a cohort of 27 progressive MS cases previously characterized on the basis of the absence/ presence of lymphoid-like aggregates in the forebrain meninges, in comparison to 11 non-neurological controls.Demyelination and meningeal inflammation of the cerebellum was greatest in those cases previously characterised as harbouring lymphoid-like structures in the forebrain regions. Meningeal inflammation was mild to moderate in cerebellar tissue blocks and no lymphoid-like structures were seen. Quantification of meningeal macrophages, CD4+, CD8+ T lymphocytes, B cells and plasma cells revealed that the density of meningeal macrophages associated with microglial activation in the grey matter, and the extent of grey matter demyelination correlated with the density of macrophages and plasma cells in the overlying meninges, and activated microglia of the parenchyma.These data suggest that chronic inflammation is widespread throughout the subarachnoid space and contributes to a more severe subpial demyelinating pathology in the cerebellum

Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases

Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state

B cell rich meningeal inflammation associates with increased spinal cord pathology in multiple sclerosis

Increased inflammation in the cerebral meninges is associated with extensive subpial cortical grey matter pathology in the forebrain and a more severe disease course in a substantial proportion of secondary progressive multiple sclerosis (SPMS) cases. It is not known whether this relationship extends to spinal cord pathology. We assessed the contribution of meningeal and parenchymal immune infiltrates to spinal cord pathology in SPMS cases characterised by the presence (F+) or absence (F-) of lymphoid-like structures in the forebrain meninges. Transverse cryosections of cervical, thoracic and lumbar cord of 22 SPMS and 5 control cases were analysed for CD20+ B cells, CD4+ and CD8+ T cells, microglia/macrophages (IBA-1+), demyelination (myelin oligodendrocyte glycoprotein+) and axon density (neurofilament-H+). Lymphoid-like structures containing follicular dendritic cell networks and dividing B cells were seen in the spinal meninges of three out of 11 F+SPMS cases. CD4+ and CD20+ cell counts were increased in F+SPMS compared to F-SPMS and controls, whilst axon loss was greatest in motor and sensory tracts of the F+SPMS cases (p<0.01). The density of CD20+ B cells of the spinal leptomeninges correlated with: CD4+ T cells and total B and T cells of the meninges; with the density of white matter perivascular CD20+ and CD4+ lymphocytes (p<0.05); with white matter lesion area (p<0.05); and the extent of axon loss (p<0.05) in F+SPMS cases only. We show that the presence of lymphoid-like structures in the forebrain is associated with a profound spinal cord pathology, and local B cell rich meningeal inflammation associates with the extent of cord pathology. Our work supports a principal role for B cells in sustaining inflammation and tissue injury throughout the CNS in the progressive disease stage

The association between neurodegeneration and local complement activation in the thalamus to progressive multiple sclerosis outcome

The extent of grey matter demyelination and neurodegeneration in the progressive multiple sclerosis (PMS) brains at post‐mortem associates with more severe disease. Regional tissue atrophy, especially affecting the cortical and deep grey matter, including the thalamus, is prognostic for poor outcomes. Microglial and complement activation are important in the pathogenesis and contribute to damaging processes that underlie tissue atrophy in PMS. We investigated the extent of pathology and innate immune activation in the thalamus in comparison to cortical grey and white matter in blocks from 21 cases of PMS and 10 matched controls. Using a digital pathology workflow, we show that the thalamus is invariably affected by demyelination and had a far higher proportion of active inflammatory lesions than forebrain cortical tissue blocks from the same cases. Lesions were larger and more frequent in the medial nuclei near the ventricular margin, whilst neuronal loss was greatest in the lateral thalamic nuclei. The extent of thalamic neuron loss was not associated with thalamic demyelination but correlated with the burden of white matter pathology in other forebrain areas (Spearman r = 0.79, p < 0.0001). Only thalamic neuronal loss, and not that seen in other forebrain cortical areas, correlated with disease duration (Spearman r = −0.58, p = 0.009) and age of death (Spearman r = −0.47, p = 0.045). Immunoreactivity for the complement pattern recognition molecule C1q, and products of complement activation (C4d, Bb and C3b) were elevated in thalamic lesions with an active inflammatory pathology. Complement regulatory protein, C1 inhibitor, was unchanged in expression. We conclude that active inflammatory demyelination, neuronal loss and local complement synthesis and activation in the thalamus, are important to the pathological and clinical disease outcomes of PMS

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues

Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis

Background: The extent of cortical pathology is an important determinant of multiple sclerosis (MS) severity. Cortical demyelination and neurodegeneration are related to inflammation of the overlying leptomeninges, a more inflammatory CSF milieu and with parenchymal microglia and astroglia activation. These are all components of the compartmentalised inflammatory response. Compartmentalised inflammation is a feature of progressive MS, which is not targeted by disease modifying therapies. Complement is differentially expressed in the MS CSF and complement, and complement receptors, are associated with demyelination and neurodegeneration. Methods: To better understand if complement activation in the leptomeninges is associated with underlying cortical demyelination, inflammation, and microglial activation, we performed a neuropathological study of progressive MS (n = 22, 14 females), neuroinflammatory (n = 8), and non-neurological disease controls (n = 10). We then quantified the relative extent of demyelination, connective tissue inflammation, complement, and complement receptor positive microglia/macrophages. Results: Complement was elevated at the leptomeninges, subpial, and within and around vessels of the cortical grey matter. The extent of complement C1q immunoreactivity correlated with connective tissue infiltrates, whilst activation products C4d, Bb, and C3b associated with grey matter demyelination, and C3a receptor 1+ and C5a receptor 1+ microglia/macrophages closely apposed C3b labelled cells. The density of C3a receptor 1+ and C5a receptor 1+ cells was increased at the expanding edge of subpial and leukocortical lesions. C5a receptor 1+ cells expressed TNFα, iNOS and contained puncta immunoreactive for proteolipid protein, neurofilament and synaptophysin, suggesting their involvement in grey matter lesion expansion. Interpretation: The presence of products of complement activation at the brain surfaces, their association with the extent of underlying pathology and increased complement anaphylatoxin receptor positive microglia/macrophages at expanding cortical grey matter lesions, could represent a target to modify compartmentalised inflammation and cortical demyelination

Digital Pathology Identifies Associations between Tissue Inflammatory Biomarkers and Multiple Sclerosis Outcomes

Background: Multiple sclerosis (MS) is a clinically heterogeneous disease underpinned by inflammatory, demyelinating and neurodegenerative processes, the extent of which varies between individuals and over the course of the disease. Recognising the clinicopathological features that most strongly associate with disease outcomes will inform future efforts at patient phenotyping. Aims: We used a digital pathology workflow, involving high-resolution image acquisition of immunostained slides and opensource software for quantification, to investigate the relationship between clinical and neuropathological features in an autopsy cohort of progressive MS. Methods: Sequential sections of frontal, cingulate and occipital cortex, thalamus, brain stem (pons) and cerebellum including dentate nucleus (n = 35 progressive MS, females = 28, males = 7; age died = 53.5 years; range 38–98 years) were immunostained for myelin (anti-MOG), neurons (anti-HuC/D) and microglia/macrophages (anti-HLA). The extent of demyelination, neurodegeneration, the presence of active and/or chronic active lesions and quantification of brain and leptomeningeal inflammation was captured by digital pathology. Results: Digital analysis of tissue sections revealed the variable extent of pathology that characterises progressive MS. Microglia/macrophage activation, if found at a higher level in a single block, was typically elevated across all sampled blocks. Compartmentalised (perivascular/leptomeningeal) inflammation was associated with age-related measures of disease severity and an earlier death. Conclusion: Digital pathology identified prognostically important clinicopathological correlations in MS. This methodology can be used to prioritise the principal pathological processes that need to be captured by future MS biomarkers

Calorie restriction activates new adult born olfactory‐bulb neurones in a ghrelin‐dependent manner but acyl‐ghrelin does not enhance subventricular zone neurogenesis

The ageing and degenerating brain show deficits in neural stem/progenitor cell (NSPC) plasticity that are accompanied by impairments in olfactory discrimination. Emerging evidence suggests that the gut hormone ghrelin plays an important role in protecting neurones, promoting synaptic plasticity and increasing hippocampal neurogenesis in the adult brain. In the present study, we investigated the role of ghrelin with respect to modulating adult subventricular zone (SVZ) NSPCs that give rise to new olfactory bulb (OB) neurones. We characterised the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHSR), using an immunohistochemical approach in GHSR‐eGFP reporter mice to show that GHSR is expressed in several regions, including the OB but not in the SVZ of the lateral ventricle. These data suggest that acyl‐ghrelin does not mediate a direct effect on NSPC in the SVZ. Consistent with these findings, treatment with acyl‐ghrelin or genetic silencing of GHSR did not alter NSPC proliferation within the SVZ. Similarly, using a bromodeoxyuridine pulse‐chase approach, we show that peripheral treatment of adult rats with acyl‐ghrelin did not increase the number of new adult‐born neurones in the granule cell layer of the OB. These data demonstrate that acyl‐ghrelin does not increase adult OB neurogenesis. Finally, we investigated whether elevating ghrelin indirectly, via calorie restriction (CR), regulated the activity of new adult‐born cells in the OB. Overnight CR induced c‐Fos expression in new adult‐born OB cells but not in developmentally born cells, whereas neuronal activity was absent following re‐feeding. These effects were not present in ghrelin−/− mice, suggesting that adult‐born cells are uniquely sensitive to changes in ghrelin mediated by fasting and re‐feeding. In summary, ghrelin does not promote neurogenesis in the SVZ and OB; however, new adult‐born OB cells are activated by CR in a ghrelin‐dependent manner