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Gene expression profiling of cathepsin D, metallothioneins-1 and -2, osteopontin, and tenascin-C in a mouse spinal cord injury model by cDNA microarray analysis

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Abstract

The purpose of this study was to use a cDNA microarray to identify new genes involved in healing of spinal cord injury. C57BL/6 mice (7–8 weeks, male) were subjected to spinal cord compression injury (SCI) at the T7/8 level (20 g, 5 min; SCI group). For the control group, mice underwent only laminectomy. Mice were killed at 1, 3 and 7 days. cDNA transcribed from mRNA was hybridized to NIA mice 15K microarrays at each time point. We found 84 genes showing significant expressional changes, including higher and lower expression levels in the SCI groups than in the control [more than 1.0 or less than −1.0 using log ratio (base 2)]. Five genes were selected for further quantitative gene expression analysis by real-time reverse transcription (RT)-PCR. For histological examination, we applied in situ hybridization and fluorescence immunohistochemistry. Cathepsin D, metallothionein-1 (MT-1), metallothionein-2 (MT-2), osteopontin (OPN), and tenascin-C were selected for quantitative and histological analysis. Microarray analysis revealed that SCI led to the up-regulation of OPN and cathepsin D expression at 7 days and also of MT-1, MT-2, and tenascin-C expression at 1 day. Tenascin-C was re-up-regulated at 7 days. These values agreed with those of real-time RT-PCR analysis. By double labeling with in situ hybridization and fluorescence immunohistochemistry, MT-1, MT-2 and tenascin-C expression was observed in neurons and glial cells at 1 day, whereas at 7 days the main MT-2 and tenascin-C expression was found in fibronectin-positive fibroblasts. The main cathepsin D and OPN expression was observed in activated macrophages/microglia at 3 and 7 days. The five genes picked up by microarray gene expression profiling were shown to exhibit temporal and spatial changes of expression after SCI. This system is potentially useful for identifying genes that are involved in the response to SCI.

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Acknowledgements

This work was supported by grants-in-aid to Katsunori Yoshinaga for scientific research from the Ministry of Education, Science and Culture of Japan. We thank Dr. Shintaro Nomura, Osaka University, Osaka, Japan, for kindly providing the plasmid containing OPN cDNA, and Drs. Masamichi Tahara and Masatoshi Komiyama, Chiba University Graduate School of Medicine, for their technical advice and useful discussions.

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  1. Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, 260-8677, Chiba, Japan

    Masayuki Hashimoto, Masao Koda, Katsunori Yoshinaga, Atsushi Murata, Masashi Yamazaki & Hideshige Moriya

  2. Department of Neurobiology, Graduate School of Medicine, Chiba University, Chiba, Japan

    Hidetoshi Ino

  3. Department of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

    Kensuke Kojima & Kan Chiba

  4. Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan

    Masao Koda & Chisato Mori

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  1. Masayuki Hashimoto
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  2. Masao Koda
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Correspondence to Masao Koda.

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Hashimoto, M., Koda, M., Ino, H. et al. Gene expression profiling of cathepsin D, metallothioneins-1 and -2, osteopontin, and tenascin-C in a mouse spinal cord injury model by cDNA microarray analysis. Acta Neuropathol 109, 165–180 (2005). https://doi.org/10.1007/s00401-004-0926-z

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  • DOI: https://doi.org/10.1007/s00401-004-0926-z

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