Journal of Neuropathology and Experimental Neurology最新文献

Primary cilia are non-motile sensory organelles that detect extracellular signals; disruptions in their functions are linked to neurodevelopmental disorders. Because cilia lengths can rapidly change in response to stressors, they are important for both plasticity and brain homeostasis. Accurate measurement of ciliary length is therefore essential but the absence of standardized methods and the variability introduced by different techniques can compromise measurement reliability and precision. To address this challenge, our study employed two distinct methods to estimate the length of primary cilia in hippocampal subregions in mice. We compared stereology-based 3D quantification, which is considered a methodological gold standard due to its unbiased sampling design and correction for tissue shrinkage, with 3D reconstruction to measure primary cilia length. 3D reconstruction imaging used a 100× oil-immersion objective. With neuronal cilia typically ∼2-10 µm long in hippocampus, a 1-µm z-step provided multiple optical sections per cilium thereby ensuring full structural visualization. Our findings show that both methods allow simple and equally precise measurements of neuronal primary cilia length in hippocampal subregions. Their strong agreement provides researchers with reliable tools for studying primary cilia on immunohistochemically stained sections and supports a consistent methodological framework for investigating cilia dynamics.

Ex vivo MRI allows for ultra-high resolution image acquisition and valuable validation when combined with histology. Postmortem brain samples may undergo long scan times without motion artifacts. Perfluoropolyethers such as Fomblin have become a popular choice of immersion liquid for their "proton free" appearance and their ability to eliminate susceptibility artifacts from the tissue-air interface but the effect of Fomblin on downstream histology experiments has not been previously studied. We exposed rat brain samples to Fomblin (n = 4) versus a control group (n = 4). Samples underwent histochemical (Nissl) and immunohistochemical staining for NeuN to assess neurons and background staining. The optical fractionator method was applied to assess total neuron counts and tissue thickness. Samples from the Fomblin group showed consistent NeuN and Nissl staining when compared to the control group. The staining color, evenness, and neuron visualization appeared unaffected with no background staining. The total neuron counts, and tissue thickness showed only slight differences in absolute numbers between groups with insufficient evidence to conclude the distribution of total neuron number or tissue thickness differed by group. These data suggest that Fomblin may not negatively impact histochemical staining, immunohistochemistry, or total neuron counts in the short term.

Cardiotrophin-like cytokine factor 1 (CLCF1) is an unfavorable factor for the prognosis of patients with glioblastoma multiforme (GBM). This research explored the functions of CLCF1 in GBM progression and the underlying regulatory mechanisms. Reverse transcription-quantitative PCR (RT-qPCR) and Western blot were used to detect the mRNA and protein levels of targeted molecules. The abilities of GBM cell lines to proliferate, migrate, and invade and undergo apoptosis and angiogenesis were analyzed by colony formation, transwell, TUNEL, and tube formation assays, respectively. Interaction between CLCF1 and SRY-box transcription factor 9 (SOX9) was verified by chromatin immunoprecipitation (ChIP)-qPCR and dual-luciferase reporter assays. The results showed that CLCF1 was upregulated in GBM. Silencing of CLCF1 suppressed the malignant phenotypes of GBM cells in vitro and the development of GBM in a xenograft tumor model. Silencing CLCF1 also reduced programmed cell death 1 ligand 1 (PD-L1) expression in GBM cells and prolonged the longevity of CD8-positive T cells in vitro. SRY-box transcription factor 9 was highly expressed in GBM and this activated CLCF1 expression as one of its transcription factors to further enhance GBM development. Thus, CLCF1 regulated by SOX9 can enhance the development and immune evasion of GBM.

Gallyas silver staining and AT8 immunostaining are frequently used to stage tau pathology in post-mortem Alzheimer disease (AD) brains. Because of differential labeling of tau aggregation types, however, these methods result in strikingly different patterns of pathology when used in adjacent sections of the same brain. How Gallyas versus AT8 staining impacts the quantification of tau pathology distribution across brain areas and affect analysis of tau and cognitive impairment is unknown. We performed a side-by-side comparison of AT8 versus Gallyas-stained hippocampal sections from 34 patients from the University of Southern California (USC) Alzheimer's Disease Research Center (ADRC). Using images of Gallyas and AT8 stained sections, we computed overall tau density in hippocampal subregions as well as manual tangle counts and compared each of them to cognitive variables like Clinical Dementia Rating and Mini Mental State Exam in the patients. We found that AT8 had a much higher density of staining overall, and the two stains had differing distributions, with increased AT8 in Brodmann area 35 and CA1. Both stains related to cognition differentially, and Gallyas density was significantly related to post-mortem interval. These findings contribute to our understanding of how tau pathology stain choice might influence the characterization of AD.

Prion diseases are rare neurodegenerative disorders that share misfolding of the normal cellular prion protein into disease-causing isoforms known as "prions" as the critical pathophysiological event. Definite diagnosis can only be achieved through neuropathological confirmation. The neuropathological features of prion disease are well described; however, some molecular subtypes are typified by characteristic neuropathological features that are subtle or absent. Prion seeding assays have excellent specificity and have considerably improved premortem diagnostic accuracy but they have reduced sensitivity for some uncommon prion disease molecular subtypes. We developed a formalin-fixed, paraffin-embedded tissue-based prion seeding assay to serve as a complementary diagnostic tool for prion diseases. Fixed brain tissue was prepared through an optimized process involving careful defacing of tissue blocks prior to sampling and then stepwise deparaffinization and homogenization. Fixed tissue homogenates are then tested in an adapted version of a diagnostic cerebrospinal fluid (CSF) prion seeding assay, which utilizes full-length recombinant hamster prion protein as substrate. Two examples illustrate the utility of the assay by confirming prion seeding in fixed brain tissue from previously neuropathologically misdiagnosed obligate carriers of 2 different prion protein gene mutations. The importance of careful tissue sampling to rigorously maintain the diagnostic specificity of this assay is also highlighted.

Changes in the peripheral metabolome, particularly in the blood, may provide biomarkers for assessing lesion severity and predicting outcomes after spinal cord injury (SCI). Using principal component analysis (PCA) and Orthogonal Partial Least Squares Discriminatory Analysis (OPLS-DA), we sought to discover how SCI severity and location acutely affect the nuclear magnetic resonance-acquired metabolome of the blood, spinal cord, and liver at 6 h post-SCI in mice. Unsupervised PCA of the spinal cord metabolome separated mild (30 kdyne) and severe (70 kdyne) contusion injury groups but did not distinguish between lesion level. However, OPLS-DA could discriminate thoracic level T2 from T9 lesions in both blood plasma (accuracy 86 ± 6%) and liver (accuracy 89 ± 5%) samples. These differences were dependent on alterations in energy metabolites (lactate and glucose), lipoproteins, and lipids. Lactate was the most discriminatory between mild and severe injury at T2, whereas overlapping valine/proline resonances were most discriminatory between injury severities at T9. Plasma lactate correlated with blood-spinal cord barrier breakdown and plasma glucose with microglial density. We propose that peripheral biofluid metabolites can serve as biomarkers of SCI severity and associated pathology at the lesion site; their predictive value is most accurate when the injury level is also considered.

Cholinergic degeneration in the nucleus basalis of Meynert (NBM) is clinically linked to cognitive impairment and gait dysfunction in Alzheimer's disease and Parkinson's disease. Modeling cholinergic degeneration in an animal model may provide powerful opportunities to study the clinical-physiological role of the NBM and lead to new therapies. We describe a procedure to inject ME20.4 Saporin, an immunotoxin that specifically binds to and depletes cholinergic neurons stereotactically into the NBM of a rhesus monkey (Macaca mulatta). A digital non-human primate brain atlas was co-registered to the brain of the monkey. A custom-designed cranial chamber was also implanted to the skull to guide the injection. The effects of the ME20.4 Saporin injections were evaluated in vivo with PET-CT using [18F]-FEOBV as a radiotracer. This approach yielded reliable spatial accuracy and successful delivery of ME20.4 Saporin into the NBM. [18F]-FEOBV PET analyses revealed reduced radiotracer uptake in the NBM. Postmortem assessment showed a reduction of ME20.4-positive cells within the NBM. No clear effects on cognitive testing were observed. This Saporin-mediated selective destruction of cholinergic neurons in the NBM, using MRI-guidance and a cranial chamber, offers a promising method to study the pathophysiology of NBM degeneration and possible therapeutic interventions.