Neuropathology and Applied Neurobiology最新文献

Aims: To standardise and automate the quantitation of human-unique neuromelanin granules in catecholamine neurons in post-mortem tissue sections from healthy individuals at different ages to understand any changes in these granules with age.

Methods: Five- to 6-μm-thick fixed and paraffin-embedded transverse midbrain tissue sections were supplied from 47 cases from three brain banks following ethical approvals. Sections were prepared and automated digital images acquired. Standardisation and automation of the quantification of neuromelanin granules was performed using the TruAI feature of the Olympus VS200 desktop platform. Comparisons between stained and unstained sections as well as correlations with age were performed.

Results: The automated platform reliably identified both stained and unstained intracellular neuromelanin granules and extracellular pigments, showing high reproducibility in measurements across laboratories using different tissue processing methods. Extraneuronal pigments were significantly smaller than intracellular neuromelanin granules. Sections processed for haematoxylin and eosin staining impacted the size and colour of both neuromelanin and the neurons containing neuromelanin. Haematoxylin made neuromelanin bluer, and the increased tissue processing made the intracellular area occupied by neuromelanin smaller in younger people. There was an increase in neuromelanin optical density and colour change (browner) with age.

Conclusions: The TruAI automated platform reliably quantifies individual neuromelanin granules in catecholamine neurons. Extraneuronal pigments are considerably smaller in size than intracellular neuromelanin, and intracellular neuromelanin changes its properties with age. The darkening and colour change of intracellular neuromelanin suggest an increase in eumelanin over time in healthy individuals. These changes can be reliably identified using the automated platform.

Gliomas, with their intricate and aggressive nature, call for a detailed visualisation of their vasculature. Traditional 2D imaging often overlooks the spatial heterogeneity of tumours. Our study overcomes this by combining tissue clearing, 3D-confocal microscopy imaging and deep learning-aided vessel extraction, achieving comprehensive 3D visualisation of glioma vasculature in intact human tissue. Specifically, we treated formalin-fixed thick human glioma tissue sections (500 μm) with OPTIClear for transparency and performed immunofluorescent labelling. Using confocal microscopy, we obtained 3D images of glioma vasculature. For vessel extraction, we employed a specialised 3D U-Net, enriched with image preprocessing and post-processing methods. In addition, we obtained 3D images of astrocytes or glioma cells, cell nuclei and vasculature with vascular basement membrane staining. Our findings indicated that OPTIClear-enabled tissue clearing yielded a holistic 3D representation of immunolabelled vessels and surrounding cells in human glioma samples. Our deep learning technique outperformed the traditional Imaris approach in terms of accuracy and efficiency in vessel extraction. Furthermore, discernible variations in vascular morphological metrics were observed between low- and high-grade gliomas, revealing the spatial heterogeneity of human glioma vessels. Analysis of other markers demonstrated differences in glioma cell morphology and vessel wall disruption across grades. In essence, our innovative blend of tissue clearing and deep learning not only enhances 3D visualisation of human glioma vasculature but also underscores morphological disparities across glioma grades, potentially influencing pathological grading, therapeutic strategies and prognostic evaluations.

Aims: Evidence suggests Alzheimer's disease (AD) patients are at increased risk of epilepsy and that seizure incidence is associated with faster cognitive decline. Previous studies indicate hyperphosphorylated tau may play a role in this disease association; however, abnormal TDP-43 and α-synuclein deposition have not been extensively examined.

Methods: Clinical and neuropathological records of AD cases over a 5-year period were retrieved from the London Neurodegenerative Diseases Brain Bank. The 114 cases were categorised into three groups: AD plus epilepsy, AD plus hippocampal sclerosis (HS) and AD only. Semi-quantitative scores for tau, TDP-43 and α-synuclein pathology within the middle temporal gyrus, hippocampus and amygdala were compared between groups.

Results: A 12% incidence of epilepsy and/or epileptic symptomology was found among the cohort. Twelve cases (11%) showed HS. No significant difference in tau pathology scores was seen between groups. However, a significantly higher score for TDP-43 was seen in AD plus epilepsy compared with AD only in the middle temporal gyrus (p = 0.004). The burden of α-synuclein pathology was increased in the amygdala of AD plus epilepsy and AD plus HS.

Conclusions: The incidence of epilepsy within this AD cohort is higher than expected within the general population (even when matched for age), and this may be associated with increased TDP-43 burden. Understanding the relationship between AD and epilepsy may highlight mechanisms of cellular damage and tissue vulnerability.

We report an infantile spinal tumour with diffuse leptomeningeal dissemination and features of a glioma with CUX1::RAF1 fusion and the DNA-methylation class, infant-type hemispheric glioma. This strange case was similar to a previously reported tumour reported in the literature, which clustered perfectly with our case. Further reports are needed to confirm the hypothesis that this neoplasm represents a novel tumour type.

Aims: Enterovirus A71 (EV-A71) can cause fatal encephalomyelitis, but the mechanisms of its spread within the central nervous system (CNS) remain unclear. This study aimed to investigate the pathways of EV-A71 dissemination after direct intracerebral inoculation and to assess the role of the murine Scavenger Receptor Class B Member 2 (mSCARB2) receptor in this process.

Methods: A mouse-adapted EV-A71 strain (MAVS) was intracerebrally inoculated into the thalamus/hypothalamus or pons/medulla of 2-week-old ICR mice. Tissues were harvested and analysed by histopathology and viral titration at 24, 48 and 72 h post-infection (hpi). The infectivity of MAVS was also tested on N1E115 mouse neuroblastoma cells.

Results: Viral antigens at the injection sites diminished over time, with restricted centrifugal inter-neuronal spread. From 48 hpi, viral antigens increased in distant motor-related neurons of the brainstem and spinal cord. There was a poor correlation between mSCARB2 expression and sites of infection; despite high mSCARB2 expression in the brain, spread was limited, while skeletal muscle, which lacks mSCARB2, showed severe infection. Direct infection of N1E115 cells was inefficient, but viral RNA transfection resulted in robust replication.

Conclusions: The findings suggest a circuitous dissemination route: viral leakage from the CNS leads to viraemia and peripheral muscle infection, followed by retrograde axonal transport back into the brainstem and spinal cord. This pathway appears to be the dominant mode of CNS invasion, independent of mSCARB2 distribution. Alternative receptor pathways likely play a critical role in EV-A71 neuropathogenesis.

We employed a novel method for collecting oral mucosal epithelial cells, an anagar-paraffin double-embedding technique for cell fixation in cytopathological processing. OMEC biopsy demonstrated a high diagnostic efficacy for NIID: H&E staining revealed inclusions in 60% (12/20) of patients, TEM confirmed them in 40% (8/20) of cases and immunofluorescence detected p62-positive aggregates in 9/20 (45%) and uN2CpolyG in 8/20 (40%) of cases, respectively. OMEC biopsy is a simple, noninvasive technique for detecting intranuclear inclusions in NIID, avoiding the need for invasive surgical procedures such as skin or labial gland biopsies.

Aims: This study aims to develop a quantitative method for assessing collagen VI expression in cell cultures, which is crucial for the diagnosis and treatment of collagen VI-related dystrophies.

Methods: We developed a combined in-cell western (ICW) and on-cell western (OCW) assay, which we have called 'collablot', to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen-hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures.

Results: Collablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls.

Conclusions: The collablot assay represents a suitable method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies.

Aims: Diagnosis of inclusion body myositis (IBM) is difficult and currently based on a combination of clinical and (immuno)histological findings. Biomarkers facilitating the diagnostic process are needed. Alpha-synuclein (αSN) aggregates are a known histological feature of IBM, but there is a lack of information on their diagnostic relevance. Furthermore, serum αSN concentrations in IBM have not been investigated.

Methods: Immunohistochemical staining for αSN was performed on 63 biopsies (19 IBM, 21 other inflammatory myopathies, 20 other myopathies and 3 healthy controls), and αSN reactive fibres were quantified. The serum concentration of αSN was determined by ELISA in 156 serum samples (11 IBM, 25 other inflammatory myopathies, 53 hereditary myopathies, 30 mitochondriopathies and 37 healthy controls).

Results: The proportion of fibres with αSN immunoreactivity was significantly higher in IBM compared to all groups (p < 0.001) and discriminated IBM against all other neuromuscular disorders with a sensitivity of 79% and a specificity of 85%, which further improved when only non-regenerating fibres were examined. In serum, αSN concentrations in IBM were generally not different from healthy controls. However, serum concentrations were inversely correlated with disease duration (r = -0.62, p = 0.04) and positively correlated with the IBM functional rating scale (r = 0.74, p = 0.01). Consequently, stratification according to these clinical parameters showed significantly lower serum αSN concentrations in late-stage, more severely affected patients.

Conclusions: αSN reactivity may serve as an additional immunohistochemical marker for IBM diagnosis. Furthermore, this study indicates that αSN serum concentrations decrease with disease duration and clinical deterioration. Therefore, serum αSN may be provisionally considered a monitoring biomarker in IBM, pending further studies.

In the study, we employ an affordable, tissue-saving, and precise simultaneous multiplex immunofluorescence method with heat-induced antibody stripping to identify and structurally analyse nigral Lewy bodies in dopaminergic neurones. Analysis of different alpha-synuclein epitopes and proteoforms reveals an almost uniform, onion-like morphology of the Lewy bodies. The N-terminal and C-terminal domains are predominantly accessible to antibody binding in the peripheral shell of the bodies.

Congenital myopathies and congenital muscular dystrophies encompass heterogeneous clinical and genetic groups of disorders characterised by muscle weakness with antenatal or early postnatal onset. These conditions are categorised according to distinctive myopathological features and causative genes. Despite advances in diagnosis through massive parallel sequencing and progress in understanding the underlying pathogenesis, many aspects of these disorders remain poorly understood. MYL1-related congenital myopathy is an ultra-rare and severe condition, associated with a deficiency of essential/alkali light myosin and impaired development of fast-twitch type II muscle fibres. This study aims to advance the understanding of the phenotype and pathogenesis of MYL1-congenital myopathy. We analysed the clinical characteristics of two individuals harbouring three novel variants in the MYL1 gene. We conducted detailed genomic analysis and extensive studies on their muscles using histological, immunohistochemical, immunofluorescence, Western Blot and electron microscopy. Both individuals showed a very severe congenital myopathy, characterised by congenital hypotonia and weakness, requiring ventilatory and nutritional assistance. Muscle biopsy revealed dystrophic-like or myopathic changes, with notable smallness of fast-twitch type II fibres, often arranged around larger type I fibres, drawing a floret pattern. These fibres expressed developmental myosin and exhibited features of aberrant myofibrillogenesis. Type I myofibres exhibited correct sarcomere alignment, but like the small fast-twitch fibres, both showed distorted cell organelles, vacuolar aggregates and membranous debris, indicating autophagic impairment. Our findings confirm that bi-allelic MYL1 variants are associated with a severe congenital myopathy, characterised by a distinctive clinical and histopathological phenotype involving impaired type II fibre development. Additionally, our study reveals a role for MYL1 in the organisation and trophism of all muscle fibre types. SUMMARY: MYL1 biallelic variants cause severe congenital myopathy with early hypotonia and type II fibre hypotrophy. Muscle biopsy shows a distinct pattern, including floret-like fibre arrangement. Findings suggest a broader role for MYL1 in fibre organisation and autophagy across muscle fibre types.