Browsing by Subject "glial cells"

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    ROLE OF BMP7 IN ACTIVATION OF RETINAL MICROGLIA
    (Office of the Vice Chancellor for Research, 2016-04-08) Davis, Garrett L.
    Introduction: The retina, structure inside the eye that converts light to electrical signals, is a multilayered neural tissue, made of neurons and glial cells. The glial cells include Müller glia, astrocytes and microglia; which provide support and maintenance to the retina and act as a surveillance system that become activated during injury and disease. We have previously shown that BMP7 is able to trigger activation of the Müller glia and astrocytes. Here we aim to determine if BMP7 is able to trigger activation of retinal microglial. Methods: Mouse retinal microglia in vitro were treated with vehicle controls or BMP7 and known microglia activators interferon gamma (INF-γ) and lipopolysaccharide (LPS). Cells were labeled with DiI following treatments. Images of cells were analyzed using ImageJ/Fiji software for morphological changes in area and number of branches. Results: Cells treated with the positive controls, INF-γ and LPS, both showed an increase in cell area in comparison to vehicle-treated microglia. However, cells treated for 3 or 24 hours with LPS showed an increase in the number of branches in addition to the increase in cell area, whereas cells treated with INF-γ for 24 hours showed fewer branch numbers in addition to an increase in cell area in comparison to control cells. Microglia treated with BMP7 for 24 hours had a larger cell area and less number of branches compared to the vehicle control. Conclusion: Morphological analysis of microglia, in vitro, indicates that BMP7 triggers their activation in a manner similar to that of INF-γ. Future studies will compare in vitro results to retinas treated with BMP7.
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    Role of Interferon Gamma in BMP7 Mediated Gliosis
    (Office of the Vice Chancellor for Research, 2016-04-08) Grosvenor, Brendan; Dharmarajan, Subramanian; Belecky-Adams, Teri
    Introduction: The retina consists of neuronal and non-neuronal cells known as glial cells. The glial cells in the retina include the Müller glia, retinal astrocytes, and microglia. Muller glia and retinal astrocytes are known to undergo gliosis, a protective response, in reaction to diseases or damaging effects such as high blood sugar concentrations or physical injury of the retina. The Belecky-Adams lab has shown that a growth factor, bone morphogenetic protein 7 (BMP7) is capable of triggering reactive gliosis indirectly, by activating secretion of interferon gamma (IFN from microglial cells. The aim of this study is to determine if fludarabine, an inhibitor of IFNγ mediated signaling, is effective in blocking activation by IFNγ in retinal astrocytes. This aim is the first step in testing the hypothesis that a blockage of IFN signaling in BMP7-treated retinas will reduce gliosis. Methods: Retinal astrocytes cells in vitro, were treated with 50 or 100μM of STAT1 inhibitor, fludarabine, for 2 hours, followed by addition of vehicle or 150ng/ml IFNγ. Protein was extracted from cells 24h after addition of vehicle or IFNγ, and quantified using BCA protein assay. Fifty micrograms of protein was loaded onto a denaturing gel and subsequently transferred to PVDF membrane and probed with an antibody against glial fibrillary acidic protein (GFAP) and -tubulin. Bands were quantitated using densitometry and GFAP normalized to the -tubulin loading control. Results: Retinal astrocytes treated with IFN but no inhibitor showed an increase in levels of GFAP, a known marker of gliosis. Cells pre-treated with fludarabine followed by IFN showed a reduction in GFAP expression in comparison to those treated with IFN alone. Conclusions: Fludarabine is effective in blocking the effects of IFNγ in retinal astrocytes. The results will allow further investigation of our hypothesis to determine if blocking IFNin BMP7-treated retinas reduces gliosis. 1Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 Mentors: Teri Belecky-Adams, Department of Biology, Indiana University- Purdue University of Indianapolis; Subramanian Dharmarajan, Department of Biology, Indiana University- Purdue University of Indianapolis Funding provided by the Undergraduate Research Opportunity Program (UROP).
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    Standardizing methods and procedures for mouse retinal flat mounts and glial cell counts
    (Office of the Vice Chancellor for Research, 2015-04-17) Anderson III, Richard; Dharmarajan, Subramanian; Belecky-Adams, Teri L.
    Introduction: The mammalian retina contains neuronal cells as well as a number of non-neuronal glial cells. The different types of glial cells include Müller glia, retinal astrocytes, and microglia. Müller glial cells and astrocytes nourish neurons and microglia act as sentinels that respond to injury or disease within the nervous system. The long-term goal of our laboratory has been to study interactions between microglia, Muller glia and astrocytes in healthy and diseased tissue. The focus of the present study was to develop a technique that would allow the laboratory to study changes in cell number in retinal flat mounts and cultures. Methods: Immunohistochemistry (IHC) was performed to fluorescently label mature murine retinal tissue. Retinal flat-mounts were stained with SOX2, a nuclear marker for glial cells or IBA1 for microglial cells, and counter-stained with Hoechst solution to label all nuclei. Pure cultures of mouse microglial cells treated with liposomal clodronate (a drug which specifically targets and ablates microglia) and vehicle were counter stained with Hoechst solution. Cell counts were performed on the images of the fluorescently labeled samples using Image-J software. Results: Convolutions were used to filter images of immunolabeled cultures and retinal flat mounts to make the images clear enough to capture cell number. The cell count assistance protocol yielded acceptable cell count results of the stained cells and determined a detectable difference in the number of clodronate treated cells versus vehicle treated control cells. The images produced of the retinal flat-mounts were analyzed to determine the percentage of SOX2 positive Müller glia in the mature murine retinal tissue. Conclusion: A modified Image J program could be used to determine cellular number in cultures and retinal flat mounts. Mentor: Teri L Belecky-Adams, Department of Biology, Indiana University-Purdue University Indianapolis