Browsing by Subject "Neuroinflammation"
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Item C1q propagates microglial activation and neurodegeneration in the visual axis following retinal ischemia/reperfusion injury(BioMed Central Ltd., 2016-03-24) Silverman, Sean M.; Kim, Byung-Jin; Howell, Garreth R.; Miller, Joselyn; John, Simon W. M.; Wordinger, Robert J.; Clark, Abbot F.BACKGROUND: C1q represents the initiating protein of the classical complement cascade, however recent findings indicate pathway independent roles such as developmental pruning of retinal ganglion cell (RGC) axons. Furthermore, chronic neuroinflammation, including increased expression of C1q and activation of microglia and astrocytes, appears to be a common finding among many neurodegenerative disease models. Here we compare the effects of a retinal ischemia/reperfusion (I/R) injury on glial activation and neurodegeneration in wild type (WT) and C1qa-deficient mice in the retina and superior colliculus (SC). Retinal I/R was induced in mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. Glial cell activation and population changes were assessed using immunofluorescence. Neuroprotection was determined using histological measurements of retinal layer thickness, RGC counts, and visual function by flash electroretinography (ERG). RESULTS: Retinal I/R injury significantly upregulated C1q expression in the retina as early as 72 h and within 7 days in the superficial SC, and was sustained as long as 28 days. Accompanying increased C1q expression was activation of microglia and astrocytes as well as a significantly increased glial population density observed in the retina and SC. Microglial activation and changes in density were completely ablated in C1qa-deficient mice, interestingly however there was no effect on astrocytes. Furthermore, loss of C1qa significantly rescued I/R-induced loss of RGCs and protected against retinal layer thinning in comparison to WT mice. ERG assessment revealed early preservation of b-wave amplitude deficits from retinal I/R injury due to C1qa-deficiency that was lost by day 28. CONCLUSIONS: Our results for the first time demonstrate the spatiotemporal changes in the neuroinflammatory response following retinal I/R injury at both local and distal sites of injury. In addition, we have shown a role for C1q as a primary mediator of microglial activation and pathological damage. This suggests developmental mechanisms of C1q may be re-engaged during injury response, modulation of which may be beneficial for neuroprotection.Item ISOLATION OF PRIMARY ASTROCYTES FROM HUMAN BRAIN TISSUE AND ASSESSMENT OF PROTOTYPICAL INFLAMMATORY RESPONSES FOR NEURODEGENERATIVE RESEARCH(2014-03) Borgmann, Kathleen R.; Tang, Lin; Ghorpade, AnujaPurpose (a): A common link in CNS disease is inflammation and the contribution of astrocyte inflammatory responses to neurodegeneration remains a focus of investigation. Non-human glial models may be limited in providing data that extrapolate directly to human neurodegenerative diseases, thus much remains to be learned in the genetically relevant context of primary human astroglial cultures. Methods (b): Here we describe the isolation and purification of primary human astrocytes from fetal brain in detail. We expand this protocol to include the assessment of astrocyte responses to inflammation through changes in cell morphology and expression of astrocyte specific markers, mitochondrial pore opening and activity, proinflammatory chemokine secretion and glutamate uptake. Results (c): Pure cultures were uniform in size and shape, and at least 95% positive for astrocyte markers. Mitochondrial pore staining revealed punctate calcein staining, which was decreased during inflammation. Upon treatment with a prototypical mediator of astrocyte inflammatory responses, interleukin (IL)-1beta, astrocyte processes became constricted; indicating a reactive astrocytic state, chemokine secretion increased significantly and the ability of astrocytes to clear glutamate was significantly impaired. Untreated cultures that demonstrated reactive phenotypes or those that failed to attain reactive states upon IL-1beta-treatment were excluded. Conclusions (d): These parameters established a framework to assess the overall purity, health, responsiveness to inflammation and thus the suitability of the culture for experimental use of primary human astrocyte cultures for neurodegenerative research.