Characteristic phosphorylation of the extracellular signal-regulated kinase pathway after kainate-induced seizures in th
Extracellular signal-regulated kinase (ERK) pathways play a crucial role in cell growth and long-lasting neuronal plasticity. Several studies have shown that phosphorylated-ERK (p-ERK) significantly increases after kainic acid (KA) administration. However
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Characteristic phosphorylation of the extracellular signal-regulated kinase pathway after kainate-induced seizures in the rat hippocampus N. Otani, H. Nawashiro, A. Yano, H. Katoh, A. Ohnuki, T. Miyazawa, and K. Shima Departments of Neurosurgery, National Defense Medical College, Tokorozawa, Japan
Summary
Material and methods
Extracellular signal-regulated kinase (ERK) pathways play a crucial role in cell growth and long-lasting neuronal plasticity . Several studies have shown that phosphorylated-ERK (p-ERK) significantly increases after kainic acid (KA) administration. However, little or no information is available about the spatial distribution of p-ERK after KA-induced seizures. We herein show that KA-induced seizures significantly increase p-ERK in both neurons and astrocytes in rat brain using Western blots and immunohistochemistry. A strong immunoreactivity for p-ERK was induced in the dentate hilar neurons and CA3 neurons 30 mins and 6 hrs after KA injection. In addition, immunoreactivity for p-ERK was seen in astrocytes 6 hrs after KA injection. 72 hrs after KA injection, all pyramidal neurons had died. These findings suggest that the ERK pathway participates in the KA-induced neurotoxicity in the rat hippocampus.
Adult male Sprague-Dawley rats weighing 150 to 200 g were used. KA dissolved in phosphate buffered saline was prepared for the induction of limbic seizures. Animals were subcutaneously injected with KA in a dose of 12 mgjkg (n = 12). For Western blot analysis, the rats were killed by decapitation under intraperitoneal anesthesia at 15, 30 min, 6, and 72 hrs after KA administration (n = 6 per time point) . For immunohistochemistry, the rats were perfused transcardially with normal saline followed by 4% buffered paraformaldehyde at the same time points (n = 6 per time point) . The brains were embedded in paraffin. Serial coronal sections (5 urn-thick] were prepared and stained with haematoxylin and eosin. The bilateral hippocampus was used for Western blot analysis. After SDS-PAGE, the protein was transferred to PVDF membranes using an electrophoretic transfer system. The membranes were incubated with a primary antibody against either polyclonal pERKlj2 antibodies (New England Biolabs, Beverly, MA , D.S.A). Immunohistochemistry was performed by the streptavidin-biotin peroxidase complex method for immunostaining by polyclonal antibodies against p-ERKlj2 (New England Biolabs). Double staining was performed with an EnVision system (DAKO, Japan) for GFAP, and a HISTOFINE SAB-PO (R) KIT (NICHIREI Co) for p-ERKlj2.
Keywords: Kainic acid; extracellular signal regulated kinase; hippocampus ; seizure.
Introduction Mitogen activated protein kinases (MAPK) are activated by phosphorylation in response to a variety of mitogenic signals . The MAPK cascades are composed of the extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 pathways. Phosphorylated-ERK (p-ERK) plays an important role in the survival, proliferation, and differentiation of various cells. Several studie
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