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Projekt Druckansicht

Konfokalmikroskop

Fachliche Zuordnung Neurowissenschaften
Förderung Förderung in 2015
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 279565665
 
Erstellungsjahr 2019

Zusammenfassung der Projektergebnisse

Within the last three years, the confocal microscope sponsored here became an essential technical tool in our institute and has been integrated in majority of our research projects beyond the proposed initial applications. Its usage includes live cell imaging, conventional fixed tissue slices as well as various thick specimens subjected to optical clearing techniques. Three major cooperative projects benefitted from the instrument. 1) The laser scanning confocal contributed significantly to our understanding of the molecular and cellular mechanisms implicated in small vessel diseases on of our main institute research focus. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited small-vessel disease, is associated with vascular aggregation of mutant Notch3 protein, dysfunction of cerebral vessels, and dementia. Imaging of thick brain slices enabled three dimensional in depth analysis of capillary vessels and other cellular components of neurovascular unit. We could demonstrate that pericytes are the first cells impacted by Notch3 aggregation preceding the opening of the blood brain barrier and microvascular dysfunction in CADASIL mouse model. In addition, we have used confocal microscopy to obtain high-resolution images of pathologic Notch3 protein aggregates in isolated brain vessels of patients affected by the hereditary stroke syndrome. We were further able to demonstrate recruitment of the HTRA1 protease to these deposits and have provided evidence for the disease relevance of this process. 2) Atherosclerosis, a chronic inflammatory disease of blood vessels is a major risk factor for recurrent ischemic stroke. Within the collaborative research program SFB 1123 ‘Atherosclerosis – Mechanisms and networks of novel therapeutic targets' We have developed a research work on atherogenesis and vascular injury to understand the relationships between atherosclerotic plaque progression and large ischemic stroke occurrences. Taking advantage of the high sensitivity and high contrast of the GaAsp detector embedded in the confocal, we could acquire images of protein enrichment in sensitive tissue such as the atherosclerotic aorta and analyze the different stages of atheroma development. We revealed the presence of osteoclastspecific Protein (e.g. Tcirg1) in macrophages of aged ApoE knockout mice fed with high-fat diet indicative of potential mineral clearance and thereby decreased plaque stability. In counterpart, we could also demonstrate that brain ischemia induces peripheral immune alterations, essentially via alarmins, humoral mediators released by the necrotic brain tissue, accelerated atherosclerosis progression in the same mouse model. 3) The laser scanning confocal in combination with the light sheet microscopy (acquired via SyNergy) was instrumental in generating and validating tools to map cellular and subcellular alterations in the brain and in the entire organism after CNS injury. As such, we took advantage of the 25x long working distance objective lens (2.5 mm; 0.95 NA) to image astrocytes in fixed thick slices (up to 1 cm) of human and mouse brain tissue after optical clearing using iDISCO. Furthermore we could assess the bio-distribution of bone marrow cells labeled with quantum dots and transplanted through tail vein injection in the brain and within the entire body. Finally we also developed automated quantification of microglia morphological-relevant changes to ongoing pathological processes, solely based on high resolution confocal images. Using recolonized germfree mice and T cell deficient mice this method allowed us to reveal differences in microglial alterations and further demonstrate the impact of gut microbiota on stroke-induced neuroinflammation. Our current investigations focus on neuronal plasticity gained from the super-resolution detector module and high resolution microscopy (objective lens 100X) to conduct in depth analyze of dendritic spines morphology and neuronal circuit reorganization over the course of stroke recovery.

Projektbezogene Publikationen (Auswahl)

 
 

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