Neuroscience
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21765
Browse
Browsing Neuroscience by Author "Ghorpade, Anuja"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
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.Item ROLE OF ALCOHOL IN PRIMARY HUMAN ASTROCYTES(2014-03) Pandey, Richa; Ghorpade, AnujaPurpose (a): The aim of this study was to investigate alcohol-mediated activation of human astrocytes and subsquential alterations in their inflammatory functions. Methods (b): Primary human astrocytes were incubated with or without alcohol at doses of 25, 50, 100 mM for 3, 5, and 7 days. Cells and culture supernatants were collected. Astrocyte morphology was examined by immunocytochemical staining for glial fibrillary acidic protein (GFAP). Expressions of TIMP-1 and pro-inflammatory cytokines including CXCL8 and CCL2 were measured by ELISA. Cell metabolic activity, proliferation, and apoptosis were analyzed by MTT assay, BrdU cell proliferation assay, and Cell Death Detection ELISAplus assay, respectively. Results (c): Alcohol exposure altered the morphology of astrocytes to a reactive phenotype as determined by GFAP immunostaining. Alcohol significantly upregulated TIMP-1 levels in dose-dependent manner (P< 0.05), with a peak at day 5 post-treatment. Moreover, alcohol treatment significantly upregulated CCL2 (P< 0.05) while CXCL8 was significantly downregulated in a dose as well as time-dependent manner (P< 0.05). In addition, alcohol exposure significantly decreased astrocytes viability (P< 0.05) and proliferation as measured by MTT and BrdU incorporation assay respectively, and significantly increased apoptosis (P< 0.05) in parallel experiments. Conclusions (d): In summary, our results suggest that alcohol may alter astrocyte inflammatory mediators and/or regulate astrocyte functions.Item TUMOR NECROSIS FACTOR-α CONFERS CYTOTOXICITY IN ASTROCYTES UNDER OXIDATIVE STRESS VIA INHIBITION OF NF-κB SIGNALING(2014-03) Singhal, Ashutosh; Ghorpade, AnujaOxidative stress and inflammation together recognized as central feature of stroke and other neurodisorders. In acute ischemic stroke, formation of H2O2 causes brain injury, which appear to be exacerbated by inflammation such as IL-1β or TNF-α produced after reperfusion. However, evidences also show that TNF-α helps in recovery and repair. Therefore, role of TNF-α is unclear. Further, it is unknown how our brain cells i.e. astrocytes are affected when oxidative stress and inflammation coexist. Here we examined the effects of H2O2 on cell survival in cultured human astrocytes co-stimulated with TNF-α or IL-1β. Data showed H2O2-treatment significantly increased astrocytes death; however, IL-1β or TNF-α-alone did not. Interestingly, co-treatment of TNF-α, but not IL-1β with non-toxic dose of H2O2 significantly increased astrocyte cell death. The toxicity of co-treatment of TNF-α and H2O2 was significantly higher than respective dose of H2O2-alone. Investigations of mechanisms revealed that H2O2 inhibited TNF-α-induced translocation of a transcription factor NF-κB to the nucleus in astrocytes thereby inhibiting cellular defense and/or survival pathways. H2O2 also decreased other TNF-a receptor associated proteins, RIP1, IκB kinases activation, thereby inhibited IκB-α degradation and NF-κB nuclear translocation. This supports the evidence of H2O2 as a modulator of pro-inflammatory signaling and explains the increased sensitivity of astrocytes during brain injury. These data also signify need to design strategy to combat oxidative stress during neuroinflammation and repair. Purpose (a): Oxidative stress and inflammation together recognized as central feature of both acute and chronic neurological disorders. In acute ischemic stroke formation of H2O2 causes brain injury, which appear to be exacerbated by IL-1β or TNF-α produced after reperfusion. However, evidences also show that TNF-α helps in recovery and repair. Therefore, role of TNF-α is unclear. Further, it is unknown how astrocytes are affected when oxidative stress and inflammation coexist. Methods (b): Here we examined the effects of H2O2 on cell survival and NF-kB dynamics in cultured human astrocytes co-stimulated with TNF-α or IL-1β. Results (c): Data showed H2O2-treatment significantly increased apoptosis in astrocytes in dose-dependent manner; however, IL-1β or TNF-α-alone did not. Interestingly, co-treatment of TNF-α, but not IL-1β with non-toxic dose of H2O2 significantly increased apoptosis in astrocytes. The toxicity of co-treatment of TNF-α and H2O2 was significantly higher than respective dose of H2O2-alone. Investigations of mechanisms revealed that H2O2 inhibited TNF-α-induced translocation of NF-kB to the nucleus in astrocytes thereby inhibiting cellular defense and/or survival pathways. H2O2 decreased TNFR1 associated protein, RIP1 level, necessary for IkB kinases activation, thereby inhibited IkB-a degradation and NF-kB nuclear translocation. The real time PCR analysis of oxidative stress pathway showed H2O2 decrease of antioxidant machinery of astrocytes. Investigation of apoptosis pathway showed that H2O2 increased the expression of TRAILR1/R2, Fas and FADD, which lead TNF-α-induced caspase-dependent apoptosis. Conclusions (d): This study supports the evidence of H2O2 as a modulator of pro-inflammatory signaling and explains the increased sensitivity of astrocytes during brain injury. These data also signify need to design strategy to combat oxidative stress during neuroinflammation and repair.