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Journal of the Korean Child Neurology Society 2003;11(1):13-19.
Published online May 30, 2003.
Organotypic Slice Culture of Rat Hippocampus for Neuroscience Research.
Soon Cheul Hong, Young Chul Youn, Dong Keun Lee, Dong Wook Kim, Soo Ahn Chae
1Department of Pediatrics, College of Medicine, Chung-Ang University, Seoul, Korea. kidbrain@korea.com
2Department of Neurology, College of Medicine, Chung-Ang University, Seoul, Korea.
3Department of Pediatrics, College of Medicine, Inje niversity, Ilsan Paik Hospital, Goyang, Korea.
Abstract
PURPOSE
Organotypic slice cultures are suitable for morphological and electrophysiological studies, and a valuable method to evaluate physiological and pathological responses to external stimuli. This study was designed to establish a method and to assess its values of organotypic slice cultures of rats' hippocampus for neuroscience research. METHODS: 8-day-old neonatal Sprague-Dawley rat's brain was dissected quickly and the brain was removed. Both of the hippocampi were dissected out in Petri dishes and placed on a chopper or tissue cutter with Gey's balanced salt solution(GBSS). Slices of 450 micrometer were cut and separated by adding of a drop of GBBS. Extra GBBS was aspirated. Transfer tissue slices of Millicell membrane were inserted in six-well plates containing 1 mL of warmed Gahwiler's media. Six-well plates were placed in an incubator at 36 degrees C with 5% CO2, and the media were changed regularly every 2 to 3 days. Some tissues were placed in 4% paraformaldehyde for staining and others were placed in Phosphate buffered saline(PBS) for 30 min for Western blotting. Then, we stained the free-floating, cryocutting or paraffin embedded slices with H&E or the days of 6, 9, 14 and 24 in vitro(DIV), and evaluated any changes of tissues and neurons by an image analysis system RESULTS: Hippocampal cultures had well-defined cell body layers of dentate gyrus, CA1, 2, 3 and 4 as early as the day of 6 in vitro(DIV). After 14 DIV, the cultures became gradually thinner from 450 micrometer to 150 micrometer. After 21 DIV, there were migrations of cells away from the margins of the slices and degenerative changes of some neuronal cells occurred. But pyramidal cells always were organized in well-defined cellular layer even after several weeks in the culture. Therefore, the best results were obtained by culturing slices of 6 to 14 DIV. Slice cutures was maintained after 4 weeks in vitro, but the oppotunity of contamination and infection increased as the periods of cultures trolonged. In staining, after any tissue was cultured in 2 weeks in vitro, no differentiation of the morphology and distribution in dentate gyrus, CA1, 2 and 3 were seen. In organo-typic slice culture of rats' hippocampus, we witnessed growth of glial and neuronal cell, and found pyramidal and granular cells. NeuN proteins were identified by Western blotting, and the density of NeuN protein bands was at the maximum value on the day of 9 in vitro. CONCLUSION: Since the organotypic slice cultures of rats' hippocampus were similar to the hippocampus in vivo in terms of anatomical and cellular morphology, it will become a valuable method for neuroscience research.
Key Words: Organotypic slice culture, Hippocampus
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