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Principal Investigator: Peter J. Syapin
Senior Associate: Janet S. Dertien
Consistent with host defense responses in the periphery, immune and inflammatory reactions in the CNS are inherently beneficial, but subject to dysregulation or inappropriate activation by pathogens and chemical mediators as occurs for other components of the innate and adaptive immune response. Thus, it is not easy to know whether an immune or inflammatory response is beneficial or detrimental. An informative example is seen for the inducible nitric-oxide synthase (iNOS). Mice lacking iNOS are susceptible to infection by Mycobacterium tuberculosis and Leishmania major, among other microbes, while they are also resistant to Influenza virus pneumonitis, LPS-induced lung damage, and other pathologies(see Nathan, 1997 for a review). Exposure to chronic high doses of ethanol can result in neurotoxicity, neuroinflammation, and changes in glial cell numbers, which are described in the sections on Background and Preliminary Studies. However, the meaning of the neuroinflammatory and glial cell responses is unclear. Using the newly generated antibody, NP155, a recent study based on animal models of Alzheimer’s and other CNS pathologies proposed that a detrimental or beneficial glial response can be inferred based on the differential expression of the NP155 antigen in either microglia or astrocytes (Ji et al., 2008). Microglia-positive and astrocyte-negative expression was detrimental, while microglia-negative and astrocyte-positive expression was beneficial. The antigen is encoded by the Tspo gene. It is an 18 kDa protein that binds PK11195 and is best known as the peripheral(-type) benzodiazepine receptor (PBR). A recent name change proposal (Papadopoulos et al., 2006) was accepted by the NCBI and the preferred name for the antigen is now “translocator protein (18 kDa)” in humans, or just “translocator protein” in rodents. To avoid confusion with the literature cited, we will refer to the antigen by its alternate designation; PBR. Previous studies have reported increased PBR in the brains of alcohol dependent mice (Syapin and Alkana, 1988) and brain damaged rats (Obernier et al., 2002b), but no cellular localization was performed. The object of this proposal is to discover, using the NP155 antibody, whether alcohol-induced brain damage elicits a beneficial or detrimental glial response based on the recent work by Ji et al. (2008). Therefore,
Specific Aim 1 is to determine the expression pattern of the PBR antigen between astrocytes and microglia during and after brain damage induced by ethanol.
Male and female rats will be exposed to ethanol by the Majchrowicz procedure and used after 1, 2, and 4 days of intoxication for immunohistochemical detection of the activated microglial marker antigen CD11b (OX42), the pan-microglial marker ionized calcium binding adapter molecule-1 (Iba-1), the reactive astrocyte marker antigen glial fibrillary acidic protein (GFAP), and PBR. Other rats will be intoxicated for 4 days and used for immunohistochemistry after 2, 8, and 30 days of withdrawal. Brain sectioned from areas of known neurodegeneration will be examined, as well as non-damaged areas with known occurrences of neuroinflammation. Rats injected with kainic acid (KA) will be used as positive controls to validate the immunohistochemical procedures. The expected result for KA-injected male rats is a nicely demarcated pattern of Iba-1 positive microglia surrounded by GFAP positive astrocytes in the midbrain, with only reactive, OX42 positive microglia expressing the PBR antigen (Ji et al., 2008).