An open-source pipeline for analyzing changes In microglial morphology

Changes in microglial morphology are powerful indicators of the inflammatory state of the brain. Here we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and “inflammation” training data and uses dimensionality reduction to generate a single metric of morphological change (an ‘inflammation index’). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.</p

[Protocol] Imaging pericytes and the regulation of cerebral blood flow

The brain’s high energy requirements drive the need for close coupling of local neuronal activity to blood supply. Capillaries have been shown to dilate before arterioles in response to sensory stimulation, pointing to a key role for microvascular pericytes in mediating cerebrovascular dynamics. However, many aspects of these cells’ function remain unknown and even controversial, from their identification, to the mechanism and regulation of their contractility in physiology and disease. Investigating how pericytes regulate vascular diameter is therefore likely to be the subject of many future experiments. Here we provide protocols for three different techniques (ex vivo slice imaging, in vivo imaging, and immunohistochemistry) that are highly valuable for performing such experiments.</p

Data for 'Risk factors for severe COVID-19 disease increase SARS CoV-2 infectivity of endothelial cells and pericytes'

Data for paper published in Open Biology (12/06/2024)Each .csv file is the data for a different figure or sub-figure, as indicated in the file name. All data is viral titre in different cells in response to incubation with SARS CoV-2 pseudo-typed virus (PV). The cells, genotype, type of PV and other experimental variables are listed in column headers.Matching R markdown code to use these data to generate the figures in the linked manuscript are provided. These can be used by saving all the data files into a folder, from which there is a subfolder called "Analysis". The R code will then generate the figures and save analyses into that subfolder.AbstractCOVID-19 was initially considered a primarily respiratory disease but is now known to affect other organs including the heart and brain. A major route by which COVID-19 impacts different organs is via the vascular system. We studied the impact of apolipoprotein E (APOE) genotype and inflammation on vascular infectivity by pseudo-typed SARS-CoV-2 viruses in mouse and human cultured endothelial cells and pericytes. Possessing the APOE4 allele or having existing systemic inflammation is known to enhance the severity of COVID-19. Using targeted replacement human APOE3 and APOE4 mice, and inflammation induced by bacterial lipopolysaccharide (LPS) we investigated infection by SARS-CoV-2. Here, we show that infectivity was higher in murine cerebrovascular pericytes compared to endothelial cells, and higher in cultures expressing APOE4. Furthermore, increasing the inflammatory state of the cells by prior incubation with LPS increased infectivity into human and mouse pericytes, and human endothelial cells. Our findings provide insights into the mechanisms underlying severe COVID-19 infection, highlighting how risk factors such as APOE4 genotype and prior inflammation may exacerbate disease severity by augmenting the virus's ability to infect vascular cells.</p

Supporting Data for Gradual Not Sudden Change: Multiple Sites of Functional Transition Across the Microvascular Bed

The data provided was used to generate the figures in Shaw et al (2022); Gradual Not Sudden Change: Multiple Sites of Functional Transition Across the Microvascular Bed, Frontiers in Aging Neuroscience. Full details of how the data was generated and processed is provided in that paper. The ReadMe file attached to this record gives details on the data including measurements and column headings.A single Excel spreadsheet containing all the data points used for graphs in Figures 4-9 and Supplementary Figures 3-6 as individual work sheets (uploaded as .xlsx), and individual .csv files containing all the data points used for graphs in Figures 4-9 and Supplementary Figures 2-6 (for non-proprietary format). Abstract In understanding the role of the neurovascular unit as both a biomarker and target for disease interventions, it is vital to appreciate how the function of different components of this unit change along the vascular tree. The cells of the neurovascular unit together perform an array of vital functions, protecting the brain from circulating toxins and infection, while providing nutrients and clearing away waste products. To do so, the brain’s microvasculature dilates to direct energy substrates to active neurons, regulates access to circulating immune cells, and promotes angiogenesis in response to decreased blood supply, as well as pulsating to help clear waste products and maintain the oxygen supply. Different parts of the cerebrovascular tree contribute differently to various aspects of these functions, and previously, it has been assumed that there are discrete types of vessel along the vascular network that mediate different functions. Another option, however, is that the multiple transitions in function that occur across the vascular network do so at many locations, such that vascular function changes gradually, rather than in sharp steps between clearly distinct vessel types. Here, by reference to new data as well as by reviewing historical and recent literature, we argue that this latter scenario is likely the case and that vascular function gradually changes across the network without clear transition points between arteriole, precapillary arteriole and capillary. This is because classically localised functions are in fact performed by wide swathes of the vasculature, and different functional markers start and stop being expressed at different points along the vascular tree. Furthermore, vascular branch points show alterations in their mural cell morphology that suggest functional specialisations irrespective of their position within the network. Together this work emphasises the need for studies to consider where transitions of different functions occur, and the importance of defining these locations, in order to better understand the vascular network and how to target it to treat disease. <br

Publisher correction: Neurovascular coupling and oxygenation are decreased in hippocampus compared to neocortex because of microvascular differences (Nature Communications, (2021), 12, 1, (3190), 10.1038/s41467-021-23508-y)

The original version of this Article contained errors in Eq. (1), in which Hbt(t) was used as a numerator and denominator instead of Hbr(t)

Risk factors for severe COVID-19 disease increase SARS-CoV-2 infectivity of endothelial cells and pericytes

COVID-19 was initially considered a primarily respiratory disease but is now known to affect other organs including the heart and brain. A major route by which COVID-19 impacts different organs is via the vascular system. We studied the impact of apolipoprotein E (APOE) genotype and inflammation on vascular infectivity by pseudo-typed SARS-CoV-2 viruses in mouse and human cultured endothelial cells and pericytes. Possessing the APOE4 allele or having existing systemic inflammation is known to enhance the severity of COVID-19. Using targeted replacement human APOE3 and APOE4 mice, and inflammation induced by bacterial lipopolysaccharide (LPS) we investigated infection by SARS-CoV-2. Here, we show that infectivity was higher in murine cerebrovascular pericytes compared to endothelial cells, and higher in cultures expressing APOE4. Furthermore, increasing the inflammatory state of the cells by prior incubation with LPS increased infectivity into human and mouse pericytes, and human endothelial cells. Our findings provide insights into the mechanisms underlying severe COVID-19 infection, highlighting how risk factors such as APOE4 genotype and prior inflammation may exacerbate disease severity by augmenting the virus's ability to infect vascular cells.</p

APOE4 expression confers a mild, persistent reduction in neurovascular function in the visual cortex and hippocampus of awake mice

Vascular factors are known to be early and important players in Alzheimer’s disease (AD) development, however the role of the ε4 allele of the Apolipoprotein (APOE) gene (a risk factor for developing AD) remains unclear. APOE4 genotype is associated with early and severe neocortical vascular deficits in anaesthetised mice, but in humans, vascular and cognitive dysfunction are focused on the hippocampal formation and appear later. How APOE4 might interact with the vasculature to confer AD risk during the preclinical phase represents a gap in existing knowledge. To avoid potential confounds of anaesthesia and to explore regions most relevant for human disease, we studied the visual cortex and hippocampus of awake APOE3 and APOE4-TR mice using 2-photon microscopy of neurons and blood vessels. We found mild vascular deficits: vascular density and functional hyperaemia were unaffected in APOE4 mice, and neuronal or vascular function did not decrease up to late middle-age. Instead, vascular responsiveness was lower, arteriole vasomotion was reduced and neuronal calcium signals during visual stimulation were increased. This suggests that, alone, APOE4 expression is not catastrophic but stably alters neurovascular physiology. We suggest this state makes APOE4 carriers more sensitive to subsequent insults such as injury or beta amyloid accumulation.</p

SARS-CoV-2 variants of concern alpha, beta, gamma and delta have extended ACE2 receptor host ranges

Following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) in PR China in late 2019 a number of variants have emerged, with two of these - alpha and delta - subsequently growing to global prevalence. One characteristic of these variants are changes within the spike protein, in particular the receptor-binding domain (RBD). From a public health perspective, these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host range of the virus. Using viral pseudotyping, we examined whether the variants of concern (VOCs) alpha, beta, gamma and delta have differing host angiotensin-converting enzyme 2 (ACE2) receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes that we subsequently attributed to N501Y and E484K substitutions within the spike RBD

A multi-disciplinary commentary on preclinical research to investigate vascular contributions to dementia

Although dementia research has been dominated by Alzheimer's disease (AD), most dementia in older people is now recognised to be due to mixed pathologies, usually combining vascular and AD brain pathology. Vascular cognitive impairment (VCI), which encompasses vascular dementia (VaD) is the second most common type of dementia. Models of VCI have been delayed by limited understanding of the underlying aetiology and pathogenesis. This review by a multidisciplinary, diverse (in terms of sex, geography and career stage), cross-institute team provides a perspective on limitations to current VCI models and recommendations for improving translation and reproducibility. We discuss reproducibility, clinical features of VCI and corresponding assessments in models, human pathology, bioinformatics approaches, and data sharing. We offer recommendations for future research, particularly focusing on small vessel disease as a main underpinning disorder.</p

Long COVID: mechanismen, risikofaktoren und genesung

Long COVID, die lang anhaltende Krankheit und Erschöpfung, die bei einem kleinen Teil der SARS-CoV-2-Infizierten auftritt, stellt eine zunehmende Belastung für die Betroffenen und die Gesellschaft dar. Eine virtuelle Tagung der Physiological Society im Februar 2022 brachte Kliniker und Forscher zusammen, um das aktuelle Verständnis der Mechanismen, Risikofaktoren und Genesung nach Long COVID zu erörtern. In dieser Übersichtsarbeit werden die Themen behandelt, die sich aus dieser Tagung ergeben haben. Die Übersichtsarbeit befasst sich mit der Natur von Long COVID, untersucht den Zusammenhang mit anderen postviralen Erkrankungen wie der myalgischen Enzephalomyelitis/dem chronischen Erschöpfungssyndrom und zeigt auf, wie die Forschung zu Long COVID helfen kann, Patienten mit allen möglichen postviralen Syndromen besser zu unterstützen. Die Forschung zu Long COVID hat besonders rasche Fortschritte bei Bevölkerungsgruppen gemacht, die ihre körperliche Leistungsfähigkeit routinemäßig überwachen, insbesondere beim Militär und bei Leistungssportlern. In der Übersichtsarbeit wird hervorgehoben, inwiefern das hohe Niveau von Diagnose, Intervention und Erfolgskontrolle in diesen aktiven Bevölkerungsgruppen Informationen über Managementstrategien für die Allgemeinbevölkerung liefern kann. Anschließend wird untersucht, wie eine Schlüsselkomponente der Leistungsüberwachung bei diesen aktiven Bevölkerungsgruppen, das kardiopulmonale Training, Long-COVID-bedingte Veränderungen in der Physiologie aufdeckt – einschließlich Veränderungen der peripheren Muskelfunktion, der ventilatorischen Ineffizienz und der autonomen Dysfunktion. Das Wesen und die Auswirkungen der Dysautonomie werden im Zusammenhang mit dem posturalen orthostatischen Tachykardiesyndrom, der Fatigue und den Behandlungsstrategien, die darauf abzielen, der Überaktivierung des Sympathikus durch Stimulation des Vagusnervs entgegenzuwirken, erörtert. Anschließend untersuchen wir die Mechanismen, die den Symptomen von Long COVID zugrunde liegen. Dabei konzentrieren wir uns auf die gestörte Sauerstoffversorgung durch Mikrokoagulation und die Störung des zellulären Energiestoffwechsels, bevor wir Behandlungsstrategien betrachten, die direkt oder indirekt auf diese Mechanismen abzielen. Dazu gehören ein fernbetreutes Atemmuskeltraining und integrierte Versorgungspfade, die Rehabilitation und medikamentöse Interventionen mit der Erforschung des Zugangs zur Long-COVID-Versorgung in verschiedenen Bevölkerungsgruppen kombinieren. Insgesamt zeigt diese Übersichtsarbeit, wie im Rahmen der physiologischen Forschung die bei Long COVID auftretenden Veränderungen aufgedeckt werden und wie verschiedene therapeutische Strategien zur Bekämpfung dieser Erkrankung entwickelt und getestet werden.</p