Minocycline is an antibiotic now recognized to have antiapoptotic and antiinflammatory properties. Because of these properties, minocycline may be of benefit in reducing neuronal apoptosis from ischemia and subsequent postischemic inflammation if administered soon after a stroke. We now explore the feasibility of using 99m Tc-annexin V, an in vivo marker of apoptosis, with SPECT to monitor the antiapoptotic effects of minocycline therapy. Methods: CB6/F1 adult male mice underwent unilateral distal middle cerebral artery occlusion (dMCA) occlusion and were imaged and sacrificed at 1, 3, 7, or 30 d after injury. Animals were given minocycline (or vehicle) 30 min and 12 h after dMCA occlusion and then given 22.5 mg/kg twice daily for up to 7 d. Before imaging, behavioral tests were performed to evaluate the neurologic function. After imaging, brains were collected for histology and assessed for the degree of apoptosis and microglial activation. Results: 99m Tc-Annexin V uptake in injured hemispheres was significantly decreased 2-to 3-fold by minocycline at all time points. Minocyline reduced infarct size as seen histologically and improved behavioral indices as late as 30 d. Infarct volume as seen histologically correlated with radiolabeled annexin V uptake seen by SPECT. In situ fluorescent microscopy demonstrated that annexin V bound primarily to neurons at 1 and 3 d, with a shift toward microglia by 7 and 30 d. Conclusion: We found that minocycline significantly reduces neuronal apoptosis and infarct size and improves neurologic outcome in mice after acute focal cortical ischemia. Apopt osis has been found to play an important role in postischemic cell death and postischemic inflammation (1,2). This observation has spawned the development of new specific antiapoptotic neuroprotective drugs (3). Minocycline is a member of the tetracycline family of antibiotics with recently recognized antiapoptotic and antiinflammatory properties. The antiapoptotic properties of minocycline appear to be due to its ability to inhibit caspase-3 (4), whereas its antiinflammatory effect appears to be due to a mechanism inhibiting p38 MAPK (MAPK is mitogen-activated protein kinase) activation in microglia (5). As such, minocycline has thus been shown to protect the brain against ischemic insults (6,7) and improve functional impairment (8). Apoptosis can be imaged indirectly with radiolabeled annexin V in animal models and humans using SPECT. Annexin V is a human protein that has a high affinity for the membrane anionic phospholipid phosphatidylserine (PS), which is selectively exposed after activation of caspase-3 (9-11). In the current study, we apply annexin V SPECT to monitor the treatment response to minocycline treatment of mice subjected to an acute unilateral distal middle cerebral arterial (dMCA) ischemic injury.
MATERIALS AND METHODS
Murine Model of Experimental StrokeCB6/F1 male mice (Jackson Laboratories), 25-30 g, were housed and treated in a humane manner in strict accordance with institutional guidelines. Ninety-nine mic...