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.