Changes of Apoptosis Inducing Factor in the Rat Brain with Ischemic Injury Induced by Middle Cerebral Artery Occlusion / 체질인류학회지

ABSTRACT

It is well known that cerebral ischemia induces apoptotic neuronal cell death along with necrosis. Apoptosis is characterized by several morphological nuclear changes including chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of members of caspase family, caspase activated DNase, and several novel proteins. A novel gene, the product of which caused chromatin condensation and DNA fragmentation, was recently identified, cloned, and designated apoptosis inducing factor (AIF). AIF is a mitochondrial flavoprotein (prosthetic group flavin adenine dinucleotide) with significant homology to plant ascorbate reductase and bacterial NADH oxidase, and is normally confined to the mitochondrial space. In a variety of different apoptosis-inducing conditions, AIF translocates through the outer mitochondrial membrane to the cytosol and the nucleus. Thus, similar to cytochrome c, AIF is a phylogenetically old, bifunctional protein with an oxidoreductase function and a second apoptogenic function. In this study, to investigate changes of AIF expression levels in the rat brain during various time periods (6, 12, 18, 24 hrs) of ischemic insults, we performed permanent middle cerebral artery (MCA) occlusion model using male SD rats and the extent of ischemic injury was measured by 2% TTC (2, 3, 5-triphenyl tetrazolium chloride) staining. There were numerous TUNEL-positive neuronal cell nuclei, which implies apoptotic cell death in the areas with ischemic insult. Western blot analysis was performed with separate samples (cytosolic fraction & mitochondrial fraction). This method was designed to investigate if the expression of AIF is really increased during ischemic injury, or the increased AIF activity is caused by its release from the mitochondrial inter-membrane space to cytosol. In the immunohistochemical study using polyclonal anti-AIF antibody revealed overall distribution of AIF in the rat brain including cerebral cortex, basal ganglia and also in hippocampus that is especially sensitive to ischemia. There was apparently increased AIF immunoreactivity in the nucleus accumbens of ischemia-induced hemisphere compared to contralateral control hemisphere. Our results seem to be the first evidence on the involvement of AIF in the apoptotic neuronal cell loss with ischemic insult and will provide some clues to the pathophysiology of neuronal apoptosis induced by ischemic insult. Further researches will reveal functional relationship between AIF and other factors involved in chromatin condensation and degradation and whether AIF itself will be useful as a caspase-independent, Bcl-2-independent death inducer.