More than 90% of the 70,000 patients resuscitated annually from cardiac arrest suffer permanent, often severe, brain injury as a sequela to the ischemia associated with the cardiac arrest. The broad, long-term objectives of our work are to understand the damage mechanisms operative in brain ischemia and reperfusion and thereby identify clinically effective therapeutic interventions to forestall the frequent occurrence of severe post-resuscitation brain injury. In those brain regions most vulnerable to damage by ischemia and reperfusion, neurons exhibit a substantial inhibition of post-ischemic protein synthesis. We propose that inhibition of protean synthesis (1) occurs at the level of formation of the initiation complex, (2) as a consequence of proteolysis and oxygen radical reaction products, (3) which lead to decreased levels or altered phosphorylation of specific translation initiation factors. We further propose that (4) translation competence during reperfusion can be protected or restored by either (a) providing the active initiation factor(s) involved (in vitro) or (b) by growth factor-mediated reversal of the reperfusion-induced altered phosphorylation of initiation factors.
The specific aims of this project are to (l) examine the effect of global brain ischemia and reperfusion on the rate of initiation of protein translation. (2) examine key eukaryotic translation initiation factors (eIFs) for ischemia or reperfusion-induced alterations in levels, phosphorylation, and activity, and tissue-map observed eIF alterations, (3) examine the effects of calcium overload or radical damage on cultured neurons with respect to the above parameters (4) examine the effects of adding purified eIFs on in vitro protein translation in homogenates obtained from normal and reperfused brains and from radical-damaged or calcium overloaded cultured neurons. and (5) studs the effects of insulin as an exogenous growth factor on translation competence and phosphorylation of eIFs in the cell culture model and in the reperfused brain. Our preliminary data indicates that after 10-min. ischemia and 90- min reperfusion (1) translation initiation is inhibited. (2) eIF-4gamma is partially fragmented, and (3) eIF-2alpha is serine phosphorylated, and during more prolonged ischemia (4) eIF-4E undergoes degradation that in preliminary immunohistochemical studies is pronounced in vulnerable hippocampal neurons. The experimental design utilizes a rat model of cardiac arrest and resuscitation to characterize the effects of ischemia and reperfusion on the eIFs that control the initiation of protein synthesis (eIF-2, eIF-2B, eIF-4E, and eIF-4gamma), and utilizes a cultured rat neuroblastoma cell line (NB104 in which neuronal differentiation can be induced) to characterize the effects of controlled radical or calcium overload insults on translation and eIFs. Insulin is a clinically relevant growth factor that has been shown to improve post-ischemic neurologic outcome and to enhance translation by increased activity of eIF-4E and eIF-2alpha, and we will study its effects on translation and eIFs in both model systems.
