Phosphatidylserine (PS) is a membrane phospholipid that is selectively redistributed to a cell's outer surface during apoptosis. PS expression can be imaged with radiolabeled annexin V, an endogenous human protein, which has a high affinity for membrane bound PS in vivo. Despite extensive study there is much that is still not fully understood about annexin V imaging. In this proposal we will to determine the sensitivity, specificity, and reproducibility of technetium-99m (99mTc)-annexin V radionuclide imaging in two models of apoptosis; the first a rodent model of unilateral middle cerebral artery (MCA) ischemic injury, the second BCL-1 tumor bearing mice undergoing chemotherapy. In the first model we will address the sensitivity, test and retest variability of 99mTc-annexin V uptake in regions of neuronal injury following unilateral mild, moderate, and severe MCA stroke in rats with and without neuroprotective therapy. We will use a dedicated small animal microSPECT (single photon emission computed tomography) system for imaging. These data will be correlated with lesion(s) size and location(s) as defined by histologic analyses to permit calculation of the sensitivity and specificity of annexin V imaging for ischemia. Test and retest variability will be determined from two serial annexin V imaging studies performed within a 24 hour period. We will then study the BCL-1 syngeneic lymphoma cell line, a line engineered to express both GFP (green fluorescent protein) and luciferase for real time direct non-invasive visualization of tumor using BLI (bioluminescence imaging). We will first define the time course of annexin V uptake in the spleen following chemotherapy in BCL-1 tumor bearing mice with microSPECT, biodistribution and autoradiographic assays in relation to serial BLI measurements of tumor burden. Next we will use fluorescent (red)-annexin V co-injected with radiotracer in combination with flow cytometry, to directly quantify the number of tumor cells that are stressed (PS positive without features of apoptosis or necrosis), apoptotic or necrotic in response to chemotherapy and correlate these results with microSPECT for calculation of sensitivity and specificity of annexin V for tumor cell apoptosis. Completion of this proposal will aid in the design and planning of clinical trials that will study the use of serial annexin V imaging as a non-invasive marker of cellular injury and therapeutic efficacy.
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