Microglia are tissue resident macrophages with a wide range of critically important functions in central nervous system development and homeostasis.In this study, we aimed to characterize the transcriptional landscape of ex vivo human microglia across different developmental ages using cells derived from pre-natal, pediatric, adolescent, and adult brain samples. We further confirmed our transcriptional observations using ELISA and RNAscope.We showed that pre-natal microglia have a distinct transcriptional and regulatory signature relative to their post-natal counterparts that includes an upregulation of phagocytic pathways. We confirmed upregulation of CD36, a positive regulator of phagocytosis, in pre-natal samples compared to adult samples in situ. Moreover, we showed adult microglia have more pro-inflammatory signature compared to microglia from other developmental ages. We indicated that adult microglia are more immune responsive by secreting increased levels of pro-inflammatory cytokines in response to LPS treatment compared to the pre-natal microglia. We further validated in situ up-regulation of IL18 and CXCR4 in human adult brain section compared to the pre-natal brain section. Finally, trajectory analysis indicated that the transcriptional signatures adopted by microglia throughout development are in response to a changing brain microenvironment and do not reflect predetermined developmental states.In all, this study provides unique insight into the development of human microglia and a useful reference for understanding microglial contribution to developmental and age-related human disease.
Anatomy education continues to evolve in health professional programs as curricula shift to competency-based models and contact hours decrease. These changes in curricula may significantly alter the learning environment for students. Importantly, changes in learning environment have been shown to impact student learning strategies and well-being. It follows, then, that an investigation of students' perceptions of the learning environment is key to understand the impact of modern anatomy curriculum alterations. The current pilot study evaluated the impact of modifying examination format on the learning environment of physical therapy students participating in a human cadaveric anatomy course. Two study cohorts of first year (entry-level) physical therapy students were invited to complete a preliminary learning environment questionnaire with 13 visual analog scale items and four short answer items. One study cohort was tested with a viva (oral) practical examination, and the other, with a bell-ringer practical examination. Analysis of quantitative items revealed two significant findings: physical therapy students in the bell-ringer cohort found it was more difficult to prepare for their examination, and that they had inadequate time to respond to questions compared to the viva cohort. Analysis of qualitative items revealed distinct themes that concerned physical therapy student learning environment specific to cadaveric anatomy. These results demonstrate that examination format can influence the learning environment of physical therapy students studying cadaveric anatomy. As a result, care needs to be taken to ensure that modernized curricula align the examination format to the method of instruction and the future application of students' knowledge in clinical practice.
Abstract Ependymal cells form a specialized brain–cerebrospinal fluid (CSF) interface and regulate local CSF microcirculation. It is becoming increasingly recognized that ependymal cells assume a reactive state in response to aging and disease, including conditions involving hypoxia, hydrocephalus, neurodegeneration, and neuroinflammation. Yet what transcriptional signatures govern these reactive states and whether this reactivity shares any similarities with classical descriptions of glial reactivity (i.e., in astrocytes) remain largely unexplored. Using single‐cell transcriptomics, we interrogated this phenomenon by directly comparing the reactive ependymal cell transcriptome to the reactive astrocyte transcriptome using a well‐established model of autoimmune‐mediated neuroinflammation (MOG 35‐55 EAE). In doing so, we unveiled core glial reactivity‐associated genes that defined the reactive ependymal cell and astrocyte response to MOG 35‐55 EAE. Interestingly, known reactive astrocyte genes from other CNS injury/disease contexts were also up‐regulated by MOG 35‐55 EAE ependymal cells, suggesting that this state may be conserved in response to a variety of pathologies. We were also able to recapitulate features of the reactive ependymal cell state acutely using a classic neuroinflammatory cocktail (IFNγ/LPS) both in vitro and in vivo. Taken together, by comparing reactive ependymal cells and astrocytes, we identified a conserved signature underlying glial reactivity that was present in several neuroinflammatory contexts. Future work will explore the mechanisms driving ependymal reactivity and assess downstream functional consequences. image
Mature oligodendrocytes form myelin sheaths that are crucial for the insulation of axons and efficient signal transmission in the central nervous system. Recent evidence has challenged the classical view of the functionally static mature oligodendrocyte and revealed a gamut of dynamic functions such as the ability to modulate neuronal circuitry and provide metabolic support to axons. Despite the recognition of potential heterogeneity in mature oligodendrocyte function, a comprehensive summary of mature oligodendrocyte diversity is lacking. We delve into early 20 th -century studies by Robertson and Río-Hortega that laid the foundation for the modern identification of regional and morphological heterogeneity in mature oligodendrocytes. Indeed, recent morphologic and functional studies call into question the long-assumed homogeneity of mature oligodendrocyte function through the identification of distinct subtypes with varying myelination preferences. Furthermore, modern molecular investigations, employing techniques such as single cell/nucleus RNA sequencing, consistently unveil at least six mature oligodendrocyte subpopulations in the human central nervous system that are highly transcriptomically diverse and vary with central nervous system region. Age and disease related mature oligodendrocyte variation denotes the impact of pathological conditions such as multiple sclerosis, Alzheimer’s disease, and psychiatric disorders. Nevertheless, caution is warranted when subclassifying mature oligodendrocytes because of the simplification needed to make conclusions about cell identity from temporally confined investigations. Future studies leveraging advanced techniques like spatial transcriptomics and single-cell proteomics promise a more nuanced understanding of mature oligodendrocyte heterogeneity. Such research avenues that precisely evaluate mature oligodendrocyte heterogeneity with care to understand the mitigating influence of species, sex, central nervous system region, age, and disease, hold promise for the development of therapeutic interventions targeting varied central nervous system pathology.
Modes of anatomical instruction (especially the need to dissect cadavers) have been contested for generations. The present narrative provides an opportunity to re-approach this age-old debate and contemplate the state of anatomical sciences education through a narrative reflection of an encounter with a donor in the cadaveric anatomy laboratory.
Abstract Autopsy-derived brain tissue analysis is vital for exploring the complex landscape of neurobiology in health and disease but processing conditions during post-mortem handling can lead to significant technical artifacts affecting data interpretation. Here, we define brain transcriptomic signatures from healthy adult human brain tissue that was snap frozen in under 1 hour (Mean: 31min, Min: 10min, Max 55min) of extraction (hereafter referred to as ~ 0 hours) and compared it to brain autopsy tissue with either typical shorter (Mean: 6 hours, Min: 4h, Max: 14h) or longer (Mean: 36 hours, Min: 17h, Max: 70h) post-mortem intervals. We found a large number of differentially expressed genes in post-mortem tissue compared to snap frozen tissue, even with the shorter post-mortem intervals. These differences allowed us to define a general “artifactual” gene signature from adult human brain autopsies that arise as a result of post-mortem processing (termed Brain Artifact Gene (BAG) Signatures). We subjected the snap frozen brain samples to different times and temperatures mimicking those typical with autopsy material to determine how these common variables influence brain gene expression. Using this approach, we discovered a set of regulated genes that we defined as “Time and Temperature Response genes Underlying Transcriptional Heterogeneity (TTRUTH)” genes. Using deep learning approaches, we then developed a model capable of modelling the extent to which individual brain autopsy samples from non-neurological disease control donors express artifactual transcripts associated with post-mortem interval time and temperature. This allowed us to assign TTRUTH scores to each individual brain autopsy sample. Moreover, using single nuclear RNA sequencing on paired samples, we identified that neuronal populations are the initial expressers of these artifactual transcripts. As tissue remains at room temperature for extended periods, oligodendrocytes emerge as the predominant cell types expressing artifactual genes. Finally, we provide an Open Science website tool for others to use to determine whether their samples are subject to similar artifacts. Using this tool, the brain autopsy research community can now assign TTRUTH scores to human brain autopsy RNAseq datasets, to provide an additional quality control measure to better standardise datasets, allow additional sample stratification across experimental groups and enhance data interpretation.
Abstract The human meninges are a dynamic tri-layered brain border that plays a key role in brain development, CSF homeostasis, immune regulation, and higher-level brain function. The meninges have also been implicated in central nervous system (CNS) pathologies such as infection, autoimmunity, and brain trauma. To understand how the meningeal microenvironment is altered under pathological conditions it is necessary to have a complete understanding of its normotypic cellular architecture and function. To date, there is no complete atlas of the normotypic adult human meninges. By surgically extracting each human meningeal layer during surgery, we generated the first layer-resolved map of all meningeal cell types via an integration of whole cell single cell RNA sequencing, multiplexed error-robust fluorescence in situ hybridization (MERFISH), and protein immunolabelling. Since fibroblasts play key roles in meningeal homeostasis yet remain less well-characterised than other meningeal cell types, we deeply phenotyped these cells in all layers. We identified 10 fibroblast subpopulations with unique predicted functions that localise to distinct neuroanatomical niches. Fibroblast interaction analysis in the dura and subarachnoid space (SAS) uncovered novel interactions with vascular cell populations mediated by insulin growth factor signaling. Together, these data serve as a comprehensive resource for future investigations of meningeal function in the healthy and diseased brain.
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