Matching Items (2)
Filtering by
- Genre: Masters Thesis
Description
Characterizing and identifying neuroinflammatory states is crucial in developing treatments for neurodegenerative diseases. Microglia, the resident immune cells of the brain, regulate inflammation and play a vital role in maintaining brain health by producing cytokines, performing phagocytosis, and inducing or reducing inflammation. These functional states can be described by specific patterns of gene expression called transcriptional programs, which are determined by the activity of a set of key transcription factors that have mostly been identified. Thus, an assay for transcription factor activity could reveal the state of the microglial cells and neuroinflammation across the brain. This research developed an assay that uses a transcription factor dependent reporter to indicate which transcriptional programs are activated in the cell when exposed to different stimuli. The prototype assay quantifies nuclear factor kappa B (NF-kB) response in cultured human cells. NF-kB is a well-characterized transcription factor associated with inflammatory pathways in most cells, including microglia. The reporter construct contains an NF-kB specific responsive element that can induce fluorescence/luminescence upon activation of the transcription factor. In an iterative refinement, a dual response fluorescent reporter was developed, which uses a secondary constitutively fluorescent reporter for built-in normalization of the responsive element for microscopy studies. With further refinement, this modular system will serve as a template for less understood transcriptional enhancers allowing for rapid, low-cost assays of neuroimmune regulators and potential in vivo applications in the study of neuroinflammation.
ContributorsLieberman, Emma (Author) / Bartelle, Benjamin B (Thesis advisor) / Plaisier, Christopher L (Committee member) / Andrews, Madeline G (Committee member) / Arizona State University (Publisher)
Created2023
Description
Of the 2.87 million traumatic brain injuries (TBI) sustained yearly in the United States, 75% are diffuse injuries. A single TBI can have acute and chronic influences on the neuroendocrine system leading to hypothalamic-pituitary-adrenal axis (HPA) dysregulation and increased affective disorders. Preliminary data indicate TBI causes neuroinflammation in the hippocampus, likely due to axonal damage, and in the paraventricular nucleus of the hypothalamus (PVN), where no axonal damage is apparent. Mechanisms regulating neuroinflammation in the PVN are unknown. Furthermore, chronic stress causes HPA dysregulation and glucocorticoid receptor (GR)-mediated neuroinflammation in the PVN. The goal of this project was to evaluate neuroinflammation in the HPA axis and determine if GR levels change at 7 days post-injury (DPI).
Adult male and female Sprague Dawley rats were subjected to midline fluid percussion injury. At 7 DPI, half of each brain was post-fixed for immunohistochemistry (IBA-1) and half biopsied for gene/protein analysis. IBA-1 staining was analyzed for microglia activation via skeleton analysis in the hypothalamus and hippocampus. Extracted RNA and protein were used to quantify mRNA expression and protein levels for GRs. Data indicate increased microglia cell number and decreased endpoints/cell and process length in the PVN of males, but not females. In the dentate gyrus, both males and females have an increased microglia cell number after TBI, but there is also an interaction between sex and injury in microglia presentation, where males exhibit a more robust effect than females. Both sexes have significant decreases of endpoints/cell and process length. In both regions, GR protein levels decreased for injured males, but in the hippocampus, GR levels increased for injured females. Data indicate that diffuse TBI causes alterations in microglia morphology and GR levels in the hypothalamus and hippocampus at 7 DPI, providing a potential mechanism for HPA axis dysregulation at a sub-acute time point.
Adult male and female Sprague Dawley rats were subjected to midline fluid percussion injury. At 7 DPI, half of each brain was post-fixed for immunohistochemistry (IBA-1) and half biopsied for gene/protein analysis. IBA-1 staining was analyzed for microglia activation via skeleton analysis in the hypothalamus and hippocampus. Extracted RNA and protein were used to quantify mRNA expression and protein levels for GRs. Data indicate increased microglia cell number and decreased endpoints/cell and process length in the PVN of males, but not females. In the dentate gyrus, both males and females have an increased microglia cell number after TBI, but there is also an interaction between sex and injury in microglia presentation, where males exhibit a more robust effect than females. Both sexes have significant decreases of endpoints/cell and process length. In both regions, GR protein levels decreased for injured males, but in the hippocampus, GR levels increased for injured females. Data indicate that diffuse TBI causes alterations in microglia morphology and GR levels in the hypothalamus and hippocampus at 7 DPI, providing a potential mechanism for HPA axis dysregulation at a sub-acute time point.
ContributorsRidgway, Samantha (Author) / Thomas, Theresa C (Thesis advisor) / Newbern, Jason (Thesis advisor) / Bimonte-Nelson, Heather A. (Committee member) / Arizona State University (Publisher)
Created2019