CELLULAR AND MOLECULAR MECHANISMS INVOLVED IN INJURY FOLLOWING GLOBAL ISCHEMIA

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Recent findings from our laboratory demonstrate that postischemic hypothermia unveils a spectrum of injury processes occurring over a markedly extended time frame following global ischemia. This paradigm will enable us to study cellular and molecular mechanisms involved in early and delayed neuronal injury following global ischemia. Preliminary data summarized in the project implicate excitotoxicity as an important process involved in the acute as well as delayed postischemic injury. The proposed protocols are targeted at identifying key events involved in this process. By using in situ hybridization, immunohistochemistry, and quantitative Northern and Western blotting, we will determine whether transient global ischemia leads to alteration in the configuration of glutamate receptor subunits, resulting in abnormally enhanced susceptibility of neurons to normally sub-lethal levels of glutamate. Postischemic hypothermia may delay this process, or unmask the development of more subtle secondary injury processes. These include a surge in extracellular glutamate or quinolinic acid, generation of oxygen radicals and/or nitric oxide, all of which can participate in the excitotoxic process during the late reperfusion period. By using microdialysis, we will assess whether a secondary surge in excitatory amino acids or hydroxyl radicals occurs during the late reperfusion phase and determine whether they are linked to the delayed injury process. By using morphological and biochemical procedures, we will ascertain whether transient global ischemia leads to delayed expression of inducible nitric oxide synthase, resulting in a surge in NO production which can be detrimental during the late reperfusion phase. Since the process of secondary neuronal damage may also involve the expression of specific genes which control the onset of cell death and the development of a slowly progressive process such as programmed cell death, we will use end-labeling in situ with DNA laddering techniques to detect whether DNA fragmentation of the apoptotic type precedes the delayed neuronal damage. We will also make use of the mRNA differential display method to characterize the expression of novel genes following ischemia, and assess the effects of postischemic hypothermia on these changes. These studies will provide new data which will enable us to understand better the role of excitotoxicity in ischemic neuronal damage. Importantly, identifying mechanisms of delayed neuronal injury following ischemia may enable us to develop a pharmacological strategy that can be employed to provide permanent protection.