This is a revised Exploratory/Developmental R21 application (1 R21 DA019836-01). The main goal of our research is to explore the effects of opiates, such as morphine, on the HIV-1-induced inflammation and cytotoxicity that takes place in the brain. We will be using a newly developed HIV-1 transgenic rat as a model system and recently developed """"""""proteomic and genomic"""""""" techniques, respectively, such as antibody microarray and DMA microarray technology, along with other cutting edge methodologies including double labeling fluorescence immunocytochemistry, and Taq-Man probe real time RT-PCR. It is well-recognized that various cytokins (such as TNF-alpha and IL-1beta) and chemokines (such as MIP-1) are induced to mediate HIV-1-induced neuroinflammation and that neurons in AIDS patients die via apoptosis and activation of the effector, such as caspase-3. This apoptosis then leads to neurodegeneration. Opiates, such as morphine, have been reported to alter the serum levels of several cytokines, including TNF-alpha, which is secreted in response to an immune challenge; and potentiate Tat- induced apoptosis of neuronal cells and astrocytes along with enhanced activation of caspase-3. We, therefore, hypothesize that exposure to morphine augments the effects of HIV-1 in deregulating the expression of cytokines and chemokines, leading to accelerated inflammation and apoptosis in the brain. The following specific aims are designed to test this hypothesis: 1) To determine morphine's effects on HIV- 1-induced inflammation by determining cytokine/chemokine expression using oligo DMA micorarray and antibody microarray; and 2) To assess morphine's effects on HIV-1-induced cytotoxicity in vivo by measuring changes in neurons (NeuN-labeled cells), astrocytes (glial fibrillary acidic protein-labeled cells), and synapses (using synaptophsin and SNAP-25 to identify synapses) in combination with phosphorylated caspase-3 immunocytochemistry and the TUNEL assay. If successful, this HIV-1 transgenic rat model and the data generated from its use may shed light on molecular and cellular mechanism(s) underlying the neurotoxicity and neuropathogenesis in HIV patients with or without morphine usage, and may lead investigators to develop research projects with a new direction or new therapeutic applications. ? ?
