This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of the project is to define the mechanisms of excitotoxic neuronal injury caused by hypothermic circulatory arrest (HCA), and to develop the means to prevent it. In Dr Baumgartner's canine survival model of HCA, replicating clinical experience during cardiac operations, dogs subjected to 2 hours of circulatory arrest at 18xC sustain a consistent neurological deficit and histological pattern of selective neuronal death. Dr Baumgartner originally showed that administration of selective glutamate receptor antagonists before and after HCA reduced the neuronal necrosis. More recently, Dr Baumgartner's group have shown that neuronal death can occur by apoptopic or necrotic mechanisms. Glutamate release after HCA results in accumulation of nitric oxide (NO), which mediates neuronal death, and that inhibition of neuronal nitric oxide synthase (NOS) reduces production of NO in the brain and prevents apoptosis The main hypothesis to be tested in the latest renewal of this project is that mitochondrial dysfunction determines the mechanism of delayed excitotoxic neuronal injury after HCA by apoptosis or necrosis, and that neuronal apoptosis can be prevented by ischemic preconditioning (IPC), achieved pharmacologically by opening ATP-dependent potassium channels on the inner mitochondrial membrane. It is further hypothesized that NO may act as a mediator both of neuronal injury and neuronal protection by IPC. In preliminary experiments, Dr Baumgartner's group have shown that diazoxide, an ATP-dependent potassium channel opener, can produce pharmacologic IPC, and that this agent can prevent apoptosis in cardiomyocytes acting on the inner mitochondrial membrane. In their canine model, diazoxide has shown near total elimination of neurological deficit following HCA, with reduction in apoptosis in select neuronal populations. They have also showed that hypoxia can activate HIF-1 with induction of i NOS and production of NO, a putative molecular pathway of the late form of IPC. The purpose of this proposal is now to extend these observations using measurements of cerebral metabolism by 1H and 31P MRSI (as well as diffusion and perfusion-weighted imaging for the detection of ischemic changes) as surrogate markers of mitochondrial dysfunction in vivo, and to correlate these measures with neuronal survival, apoptosis and necrosis. These measurements will be made both in untreated HCA, and in animals with pharmacologic IPC with diazoxide. The proposed resource will interact with this project in a service capacity, since the required techniques (perfusion (and diffusion) imaging - TRD 1, proton and phosphorus MRSI - TRD 2) are already developed and available. Customized data acquisition protocols for perfusion and diffusion MRI, and for 1H and 31P MRSI in canine will be implemented for this project. The wide bore, short length and open design of the KKI magnet is particularly well suited for this particular large animal preparation which requires careful physiological monitoring during anesthesia.
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