Detection and partial purification of ischaemia-related neurotrophic activity in the periinfarcted brain tissue.

Abstract

In the rat model of middle cerebral artery (MCA) occlusion, axons originating from the ipsilateral cortical and thalamic neurons are injured by ischaemia. The cortical neurons survive thereafter without retrograde degeneration, but thalamic neurons slowly die because of retrograde degeneration. The fate of these two neurons is remarkably different and may be related to neurotrophic activity induced by ischaemia. We detected ischaemia-related neurotrophic activity, and partially purified the factor. Tissue samples were obtained from the cortex adjacent to the infarction and contralateral corresponding site at 4, 8 and 12 days after occlusion of the MCA. They were homogenated with a culture medium and ultracentrifuged. The supernatant was obtained and used for neurotrophic assay. Foetal cortical neurons were obtained from 17 days rat embryo and cultured. Neurotrophic activity was assayed by applying tissue extract to the culture medium. Application of periischaemic cortical extract obtained at 8 and 12 days after ischaemia improved neuronal survival by 50% and 200% as compared to contralateral cortical extract, respectively. The activity was not detectable at 4 days after ischaemia. The neurotrophic activity disappeared by heating the extract at 90 degrees C for 10 min. We fractionated the extract by saturated ammonium sulphate precipitation, followed by gel-filtered with Superose 12 column. The neurotrophic activity was detected in the precipitation of 30 to 60% saturation fraction of ammonium sulphate. With gel-filtration we separated neurotrophic activity in several fractions, which included marker proteins of 8, 22 and 30 kilodaltons. The activities were only detected in the lesioned side but not in the contralateral side.(ABSTRACT TRUNCATED AT 250 WORDS)