| In damaged regions, cascades of damage had occurred at early stage of cerebral ischemia/reperfusion, a series of sub-cellular and molecular pathologic changes had also taken place in cells of damaged regions, which could make cell membranae, cell organs and DNA damaged. The damages of DNA might be the result of many factors as well as be the reason of many new changes and damages in cell. At present, we knew less about DNA damages, expression change of DNA repair enzyme and its influence to DNA damages at early stage of reperfusion. This experiment was based on these considerations, and we planed to use Nissl stain method, H&E stain method, Klenow fragment of DNA polymerase I -mediated nick end labeling method (Klenow method), immunohistochemical and immunofluorescent technology, to discuss DNA damages, expression changes of DNA repair enzyme apurinic/apyrinidinic endonuclease/ redox factor 1 (APE/Ref-1) and production of immediate early genes (lEGs) c-Fos at early stage of focal cerebral ischemia/reperfusion.hi the experiment, we made rat middle cerebral artery occlusion/ reperfusion (MCAO/R), and divided healthy male SD rats into groups as follow: normal control, sham-operation and MCAO 2 hours reperfusion 2, 6, 12, 24, 48 and 72 hours by random. At each time point, we observed morphological changes, in situ detected DNA breaks, and observed expression changes of production of lEGs c-Fos and DNA repair enzyme APE/Ref-1 which participated in many kinds of DNA repairs. Results in the experiment were shown as follow:(DHE stain indicates: The model of rat MCAO/R was credible, and the damaged regions included cortex (lobus frontalis, lobus arietalis, lobus occipitalis et al) and caudatum. Profiles of Cells were right in sham-operation and normal control groups, the nucleus and nucleolus were clear. 6 hours after reperfusion, some cells in damaged regionsshowed morphological changes evidently, cell bodies began to show swollen or shrunken. 24 hours after reperfusion, lots of cells showed morphological changes, 72 hours after reperfusion, the number of cells in damaged regions decreased remarkably.(2)Nissl stain indicates: Profiles of Neurons were right in sham-operation and normal control groups, and Nissi bodies were clear. 6 hours after reperfusion, some neurons in damaged regions showed morphological changes evidently, Nissl bodies began to disappear, and neuron bodies showed swollen or shrunken. 24 hours after reperfusion, lots of neurons showed morphological changes, 72 hours after reperfusion, the number of neurons in damaged regions decreased remarkably.(3)Klenow method in situ detection indicates: No Klenow positive cells existed in sham-operation and normal control groups. 6 and 12 hours after reperfusion, a few Klenow positive cells were detected in damaged cortex and caudatum (P<0.01). 24 hours after reperfusion the number of Klenow positive cells immediately and remarkably increased (P<0.01). 72 hours after reperfusion, Klenow positive cells were less than those in 24 hour (P<0.01).(4) APE/Ref-1 immunohistichemistry and immune-fluorescent indicates: In sham-operation and normal control groups, APE/Ref-1 expressed in neuron and astrocyte nucleus in lobus frontalis, lobus arietalis, lobus occipitalis and caudatum at both sides. From 2 hours after reperfusion, the APE/Ref-1 positive cells in the regions were less than those in sham-operation and normal control groups (PO.01), and had no differences among the operation groups (P>0.05) till 48 hours. 72 hours after reperfusion, compared with other operation groups, positive cells remarkably decreased (PO.Ol). From 2 to 48 hours, APE/Ref-1 appeared in cytoplasm of cells in the cortex regions at damaged side. 72 hours after reperfusion, cells number decreased in the regions, and APE/Ref-1 positive cells decreased greatly too.(5)c-Fos immunohistichemistry indicates: In sham-operation and normal control groups, c-Fos expressed in nucleus in lobus frontalis, lobus arietalis, lobus occipitalis and caudatum at both sides. From 2 hours after r... |
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