MKP-1 MRNA AND PROTEIN EXPRESSIONS ARE INDUCED IN NEURON AT ACUTE PHASE AFTER FOCAL CEREBRAL ISCHEMIA

MKP-1 MRNA AND PROTEIN EXPRESSIONS ARE INDUCED IN NEURON AT ACUTE PHASE AFTER FOCAL CEREBRAL ISCHEMIA

H. Horikawa¹, H. Imai¹, Y. Chen(陈毅力)¹, T. Ochi¹, A. Ito¹, H. Kanemitsu², H. Nakatomi¹, N. Saito¹浙江大学医学院附属邵逸夫医院神经外科陈毅力

1Department of Neurosurgery, The University of Tokyo, 2Department of Radiological

Technology, Faculty of Medical Technology, Teikyo University, Tokyo, Japan

Objectives: Ischemia-induced changes in gene and protein expression may provide important

information relating to mechanisms of injury and potential recovery. Cells in the peri-infarct zone (penumbra) display a complex response including selectively increased mRNA levels of genes associated with stress, apoptosis, transcription, and inflammation. The study previously reported in our department by using the DNA microarray technique revealed the expression level of 246 transcripts were increased after ischemia Of them, mitogen-activated protein kinase phosphatase (MKP-1) are known to show the 10-fold increase after ischemia. The MKP-1 gene encodes a dual specificity (Tyr/Thr) protein phosphatase that specifically inactivates mitogenactivated protein (MAP) kinase, and it seems to play an important role in controlling cellular proliferation, apoptosis and stress response. However, the function of MKP-1 in brain still remains to be unknown. We examined the expression pattern of MKP-1 mRNA and protein in the brain using in situ hybridization and immunohistochemistry at several time points after focal cerebral ischemia.

Methods: Permanent middle cerebral artery occlusion model was employed in Sprague Dawley rats (300-350g, n= 30). Rats were decapitated at 30minutes, 1hr (hour), 3hrs, 6hrs, 12hrs, 24hrs, 3days, and 7days after ischemia, and sham-operation without arterial occlusion. Sections of 20μm thickness were cut. For in situ hybridization, the sections were applied by Digoxigeninlabeled cRNA for MKP-1 probes. For immunohistochemical analysis, the specific antibody for MKP-1 was applied for the adjacent sections of in situ hybridization. The antibody against NeuN for neurons was applied for double immunohistochemistry.

Results: Histopathology showed ischemic damage was identified consistently in the ipsilateral MCA territory including the frontal cortex, dorsal parietal cortex, and the caudate. In situ hybridization showed the expression of MKP-1 mRNA emerged in the peri-infarct zone and remote cortex of ischemic side of cerebral hemisphere at 30 minutes after MCA occlusion, instead of the low expression in ischemic core. This expression of MKP-1 mRNA in these regions dramatically increased at 1 hour after MCA occlusion and was maintained until 3 hours after MCA occlusion. However, MKP-1 mRNA expression in the ischemic side of cerebral hemisphere was reduced markedly at 12 hours after MCA occlusion and almost lost at 24 hours. MKP-1 mRNA expressing cells were distributed predominantly in the ipsilateral cortex, with most cells displaying neuronal morphology. The double immunohistochemistry revealed the most MKP-1 positive cells in the peri-infarct cortex in the acute phase expressed NeuN and were thus identified as neurons.

Conclusions: The major observations were that MKP-1 mRNA was induced and strongly expressed in neurons of in the peri-infarct zone and remote cortex of ischemic side of cerebral hemisphere only at acute phase and that the expression pattern was very specific and sensitive to the ischemic insult after MCA occlusion

XXVth International Symposium on Cerebral Blood Flow and Metabolism & The Xth International Conference on Quantification of Brain Function with PET （Brain2011）的会议论文