1,721,056 research outputs found
Transcriptomic heterogeneity of microglia in relation to Alzheimer's disease-associated pathology and post-mortem delay
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The role of glial calcium changes in animal models of stroke
Full text linkIschemic stroke represents one of the leading causes of death and disability. A main characteristic of the early phase of stroke is the spontaneous appearance of peri-infarct depolarizations (PIDs), which are known to negatively affect infarct size and clinical outcome. The aim of this work was to better define the dynamics of PIDs in the peri-infarct region (penumbra), and to investigate the underlying cellular pathways, with a particular emphasis on calcium signaling in astrocytes. I combined the permanent middle cerebral artery occlusion (pMCAO) stroke model with in vivo two-photon (2-P) microscopy in transgenic mice expressing cyan fluorescent protein (CFP) selectively in astrocytes under the Cx43 promoter. Calcium dynamics and local changes in cerebral blood flow (CBF) were measured using the pharmacological calcium indicator Oregon Green 488 BAPTA-1 AM (OGB-1), and blood vessels were visualized by intravenous injection of Texas Red 70 kDa dextran. In addition, astroglial edema – i. e. volume changes in astrocytes during PIDs – was estimated by measuring somatic CFP fluorescence over time. Following pMCAO induction, anesthetized mice were imaged through a cranial window. I found that PIDs could be detected by this technique based on the appearance of a typical propagating wave of calcium changes in neurons and astrocytes that was accompanied by characteristic CBF changes and the development of astrocytic edema. I confirmed the propagation pattern as well as the typical electrophysiological characteristics of PIDs in separate groups by combining pMCAO with extracellular measurements of the direct current (DC) potential and with regional CBF measurements using laser speckle contrast imaging. In addition, the calcium changes in astrocytes during PID were confirmed using a mouse line that expresses the genetically encoded calcium indicator GCaMP3 in astrocytes using a Cre/loxP approach (GLAST-CreERT2:Ai38 line). Subsequently, I aimed to identify the astroglial signaling pathways underlying calcium transients during PIDs, and their contribution to PID threshold and propagation. To this end, I induced pMCAO in transgenic mouse lines with deletions of inositol triphosphate receptor 2 (IP3R2), transient receptor vanilloid 4 (TRPV4) channel, or aquaporin-4 (AQP4). Each line was crossbred to Cx43 CFP mice for astrocyte identification. In addition, I tested the effect of pharmacological antagonists of these channels in wildtype mice as well as blockade of the astrocytic sodium calcium exchanger (NCX). I found that the amplitude of astroglial calcium changes during PID was significantly reduced in IP3R2-deficient mice. Calcium transients were also reduced in TRPV4-deficient mice and wildtype mice treated with TRPV4 or NCX blockers. These results indicate that TRPV4 and NCX channels mediate an influx of calcium from the extracellular space, and a substantial calcium release from the endoplasmic reticulum through IP3R2. Surprisingly, no changes were seen in AQP4-deficient mice. Moreover, I found that the delay between pMCAO onset and the first PID, as well as the frequency of subsequent PIDs, was significantly reduced in IP3R2-deficient mice. This was confirmed in separate groups using DC-potential recordings and laser speckle contrast imaging. In conclusion, I have found that astrocyte-specific IP3R2 is an important contributor to PID threshold, and that IP3R2, TRPV4, and NCX contribute to calcium influx during PID. Since PIDs and cellular calcium overload are important determinants of stroke severity and outcome, pharmacological modulation of these channels in translational or clinical studies may prove therapeutically beneficial
Entwicklung und Validierung eines Screeninginstrumentes zur Diagnose einer Sepsis nach Schlaganfall
Full text linkSchlaganfall-assoziierte Infektionen können eine Sepsis hervorrufen, die mit einem schlechten klinischen Outcome assoziiert ist. Die lebensbedrohliche Organdysfunktion, welche die Sepsis definiert, kann mittels Sequential Organ Failure Assessment (SOFA) Score identifiziert werden. Allerdings ist die Anwendbarkeit des SOFA-Scores bei Patient*innen außerhalb der Intensivstation eingeschränkt. Diese Arbeit beschäftigt sich damit, einen modifizierten SOFA-Score zur Vorhersage eines schlechten Outcomes und somit der Diagnose einer Sepsis bei Schlaganfallpatient*innen mit Infektion zu entwickeln und zu validieren.
Anhand einer bereits charakterisierten Kohorte von 212 Patient*innen mit Schlaganfall-assoziierter Infektion nach mechanischer Thrombektomie wurden geeignete Prädiktoren für ein schlechtes Outcome mittels logistischer Regression identifiziert und mit Hilfe des Youden-Index dichotomisiert. Zur Auswahl des finalen Modells wurden verschiedene Kombinationen dieser Prädiktoren auf die Anpassungsgüte getestet. Der daraus resultierende Score wurde anschließend an einer separaten Kohorte von 391 konsekutiven Patient*innen mit ischämischem Schlaganfall und Infektion, die im Zeitraum vom 01.01.2021 bis 30.06.2022 am Universitätsklinikum Bonn behandelt wurden, validiert.
Folgende fünf Prädiktoren wurden in den abgeleiteten Stroke-(S-)SOFA-Score eingeschlossen: National Institutes of Health Stroke Scale ≥ 14, pulsoxymetrisch gemessene Sauerstoffsättigung 9/l, Kreatinin ≥ 1,2 mg/dl. Die Fläche unter der ROC-Kurve (AUC) des S-SOFA-Scores für die Vorhersage eines schlechten Outcomes, das auf eine Sepsis hindeutet, betrug 0,713 [95 %-Konfidenzintervall: 0,665–0,762] und war damit ähnlich der des Standard-SOFA-Scores (0,750 [0,703–0,798]), jedoch wurde die vorab festgelegte Nichtunterlegenheitsgrenze von -0,06 knapp verfehlt (untere Grenze des 95 %-Konfidenzintervalls für die AUC-Differenz -0,075). Bei Patient*innen außerhalb der Intensivstation hingegen war der S-SOFA-Score dem SOFA-Score nicht unterlegen (untere Grenze des 95 %-Konfidenzintervalls für die AUC-Differenz -0,055).
Hieraus lässt sich schlussfolgern, dass der S-SOFA-Score zukünftig zur Identifikation von nicht intensivpflichtigen Schlaganfallpatient*innen mit Sepsis und hohem Risiko eines schlechten Outcomes beitragen könnte. Der Vorteil des S-SOFA-Scores gegenüber dem Standard-SOFA-Score besteht darin, dass dieser einfach anzuwenden ist und routinemäßig erhobene Parameter verwendet. Bei Intensivpatient*innen sollte der SOFA-Score weiterhin der Standard bleiben. Um unsere Ergebnisse zu bestätigen, sind prospektive multizentrische Studien erforderlich
3D culture conditions instruct an in vivo-like phenotype in primary microglia
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Deletion of <em>Cyfip1</em> in mouse microglia affects microglial actin dynamics, motility and functions, unveiling a dendritic spine maturation defect
Full text linkMicroglia are the innate immune cells in the CNS, serving as the first line of defense in the brain parenchyma. During mouse embryogenesis, microglial progenitor cells originate from the yolk sac and migrate into the CNS via the circulatory system. As the developmental stage progresses, microglia develop ramified and motile processes which constantly survey the brain parenchyma. The high motility allows microglia to respond to insults by migrating to the affected sites, and clear up the pathogens and damage. Moreover, microglia also interact with neuronal synapses with their processes. Microglial motility is driven by the intracellular force generated by actin cytoskeleton rearrangement. Essential to this process is the cytoplasmic fragile-X mental messenger ribonucleoprotein (FMRP) interacting protein 1 (CYFIP1), an essential component of the WAVE regulatory complex (WRC), which activates the ARP2/3 complex to regulate branched actin networks.
In this study, we show that CYFIP1 is the sole CYFIP isoform in microglia, and is highly expressed compared to the other cell types in the CNS. We determine that microglia possess a unique WRC, which is composed of CYFIP1, HEM1, WAVE2 and ABI3, and that depleting CYFIP1 leads to the loss of all other WRC components. We then show that in the absence of CYFIP1, microglial branching is reduced and microglial homeostasis is impaired in the mouse brain. We also, for the first time, identify the molecular role of the CYFIP1-WRC in microglial actin dynamics, as a regulator of the dynamic filamentous actin formation, which is involved in microglial phagocytosis, migration and membrane ruffling. Indeed, the absence of the CYFIP1-WRC disrupted lamellipodium formation and impaired microglial directional migration. Interestingly, in the brain of Cyfip1 conditional knockout mice, there was an unexpected alteration in dendritic spine maturation in layer V pyramidal neurons of the mouse cortex.
In summary, in this study we demonstrate the pivotal role of the CYFIP1-WRC in microglial homeostasis, motility and phagocytic function. Exploring the role of CYFIP1-dependent actin dynamics on the interaction of microglia with synapses and its significance for the establishment of neuronal circuits are intriguing topics for future research
Analysen der Akuttherapie und Komplikationen von ischämischen Schlaganfällen
Full text linkDer Schlaganfall ist weltweit eine der führenden Ursachen für Mortalität und erworbene Behinderungen. Das klinische Outcome nach einem Schlaganfall kann mittels Behandlung auf der Stroke Unit einschließlich der Akuttherapie mit intravenöser Thrombolyse und endovaskulärer Therapie positiv beeinflusst werden. In den vier Arbeiten, die dieser kumulativen Habilitationsschrift zugrunde liegen, wurden verschiedene Aspekte der Akuttherapie und der Behandlung früher Komplikationen von Schlaganfällen mittels klinischer Kohorten- und Registerstudien analysiert.
Eine Analyse des Arbeitsablaufes bei der endovaskulären Schlaganfalltherapie ergab, dass eine initiale Bildgebung mittels MRT im Vergleich zu einer CT-Bildgebung zu einer Verzögerung des Zeitintervalls von der Aufnahme bis zum Beginn der Bildgebung führt. Die Wahl der Bildgebungsmodalität beeinflusste jedoch weder die darauffolgenden Arbeitsschritte noch das klinische Outcome nach drei Monaten. Dies spricht dafür, dass sowohl eine MRT- als auch eine CT-Diagnostik eine effektive endovaskuläre Schlaganfalltherapie ermöglichen können. Bei Einsatz des MRTs ist jedoch eine Optimierung des Arbeitsablaufes, insbesondere von der Aufnahme bis zum Beginn der Bildgebung, nötig, um Verzögerungen bei der Behandlung zu vermeiden.
Das Vorliegen von rezenten Hirninfarkten gilt als Kontraindikation für die intravenöse Thrombolysetherapie bei akutem Hirninfarkt. Eine Analyse von Blutungskomplikationen und klinischem Outcome nach intravenöser Thrombolyse von Patienten, die neben dem akuten Hirninfarkt zusätzlich im MRT rezente klinische stumme Hirninfarkte aufwiesen, ergab jedoch, dass diese rezenten klinische stummen Infarkte weder mit einem erhöhten Blutungsrisiko noch einem schlechteren klinische Outcome einhergehen. Diese Ergebnisse sind hilfreich, um die Indikation zu intravenösen Thrombolysetherapie präziser zu stellen und diese Therapie einer breiteren Patientengruppe anzubieten.
Als eine wesentliche Ursache für die schlaganfallassoziierte Komplikation Fieber wurde eine schwergradige Dysphagie identifiziert. Dieser Zusammenhang wurde erstmalig anhand einer Stufendiagnostik der Dysphagie mit klinischer Schluckuntersuchung ergänzt durch apparative Verfahren belegt. Eine detaillierte Dysphagiediagnostik erscheint daher hilfreich, um Fieber als Komplikation nach Schlaganfall vorzubeugen und so ein besseres klinisches Outcome zu erreichen.
Infolge schlaganfallassoziierter Infektionen kann es zu einer Sepsis kommen. In der Analyse einer Kohorte von Schlaganfallpatienten mit Verschluss eines großen hirnversorgenden Gefäßes war festzustellen, dass die Sepsis eine häufige Komplikation ist und einen unabhängigen Prädiktor für ein schlechtes klinisches Outcome darstellt. Zusammenfassend bieten die Ergebnisse der vier Arbeiten, die dieser kumulativen
Habilitationsschrift zugrunde liegen, mehrere Ansatzpunkte, mit denen die Akuttherapie von Schlaganfällen verbessert und frühe schlaganfallassoziierte Komplikationen vermieden und behandelt werden können
Sphingosine-1-phosphate receptor 3 signaling is a critical modulator of stroke outcome
Full text linkStroke is a leading cause of long-term disability worldwide. Its highly complex pathogenesis is characterized by a deleterious cycle of vascular dysfunction and inflammation. Recently, the bioactive phospholipid sphingosine-1-phosphate (S1P), has gained increasing attention in cardiovascular diseases due to its involvement in both vascular function and immune cell responses. Altered S1P levels have been reported in several cardiovascular and inflammation-associated diseases, including stroke. S1P signals via five ubiquitously expressed S1P receptors, S1Pr1-5. Previous in vitro studies revealed alterations of S1Pr3 signaling under inflammatory conditions. During vascular adaptation to disease models of middle cerebral artery occlusion (MCAo), we determined the expression pattern of S1Pr3 in brain tissue with qPCR and western blot. Transient MCAo in endothelial- and astrocyte-specific RiboTag transgenic mice allowed us to determine changes in S1P signaling through immunoprecipitation of translating mRNAs from both cell types. For modulation of S1Pr3 signaling, we used S1Pr3-/- mice or pharmacological S1Pr3 inhibition administered 4 and 8 hours after permanent MCAo. Stroke outcome was determined by infarct size evaluation, neuroscore, and assessment of cerebral blood flow (CBF) using magnetic resonance imaging.
S1Pr3 expression was significantly increased 1 day and 3 days post-ischemia in the ipsilateral hemisphere of WT mice on the gene and protein level. Mice lacking S1Pr3 revealed improved neurological function and reduced ischemic lesion during the acute phase after experimental stroke. Using vessel-parenchyma fractionation of brain tissue, we detected the majority of S1Pr3 associated with cerebral vessels. RiboTag analysis unveiled an augmentation of astrocyte- but not endothelial-specific S1Pr3 expression 1 day and 3 days post-stroke. RiboTag results were further confirmed using in situ hybridization colocalization of Gfap and Sox9, astrocytic markers and S1Pr3 in the ischemic hemisphere. Single administration of an S1Pr3 antagonist 4 hours after permanent MCAo led to significant CBF improvements in the ipsilateral hemisphere 1 day post-stroke that persisted up to 3 days. Consequently, infarct size was significantly reduced in mice treated with S1Pr3 antagonist. However, later administration at 8 hours post-stroke did not improve CBF or reduced infarct size.
In conclusion, our findings point to an important involvement of the S1P/S1Pr3 signaling axis during stroke, and a potential contribution of astrocytes-specific S1Pr3 signaling during the acute phase post-stroke. Modulating S1Pr3-mediated vascular responses may emerge as a viable target to improving stroke outcome
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Impact of Microglial CYFIP1 Deletion on Astrocytes, Oligodendrocytes, and Myelination in a conditional mouse model
No full textCYFIP1 is one of the five subunits of the WAVE regulatory complex, which is involved in controlling actin cytoskeleton remodeling, impacting many cell processes including cell motility (Eden et al., 2002; Rottner et al., 2021). A CYFIP1 dysregulation is associated with multiple neurological and psychiatric disorders. For example, a reduction of CYFIP1 is prevalent in patients with schizophrenia (Sayad et al., 2018; Stefansson et al., 2008).
In this project, I used a conditional mouse model, in which CYFIP1 is deleted in myeloid cells including microglia, to study the effect of microglial CYFIP1 on astrocytes and oligodendrocytes in the murine CNS. I found a significant upregulation of GFAP in astrocytes, using immunofluorescence analysis of cerebral sections from young adult CYFIP1 KO mice, along with a decrease in astrocytic cell number, as quantified by the number of SOX-9-positive cells.
In addition, I examined oligodendrocyte cell number, and myelination by immunofluorescence labeling of MBP, OLIG2, and APC in cerebral sections of the same mouse line. Here, I found a decrease in myelination in the corpus callosum, and a decrease in oligodendrocytes in the somatosensory cortex, as well as the corpus callosum.
To better understand the mechanisms and to test whether soluble factors from CYFIP1 depleted microglia induce the altered astrocytic phenotype, I performed a set of experiments using astrocyte cultures, which were incubated with microglia-conditioned medium from cultured control vs. CYFIP1 KO microglia. To this end, I analyzed astrocytic phagocytosis with a functional assay, and a defined set of genes including synaptogenic genes, phagocytic genes, and interferon-stimulated genes by qPCR. There were no obvious results from these cell culture experiments that would explain the upregulation of GFAP or reduced astrocyte number that I detected in the conditional CYFIP1 mouse model, as I detected no decrease in cell number or proliferation of cultured astrocytes. However, there was a significant decrease of Mtor, the gene encoding mTOR, which has been shown to upregulate cell proliferation, and which may cause a decreased proliferation of astrocytes. Further experiments using astrocyte-microglia co-cultures and additional analyses in conditional CYFIP1 KO mice will be needed to elucidate potential pathways that underlie the changes in astrocytes and oligodendrocytes associated with conditional myeloid cell-specific CYFIP1 deletion
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