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.
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.
Hashimoto
Masayuki
M
Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, 260-8677, Chiba, Japan.
Koda
Masao
M
Ino
Hidetoshi
H
Yoshinaga
Katsunori
K
Murata
Atsushi
A
Yamazaki
Masashi
M
Kojima
Kensuke
K
Chiba
Kan
K
Mori
Chisato
C
Moriya
Hideshige
H
D000818:Animals; D001522:Behavior, Animal; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D016000:Cluster Analysis; D005455:Fluorescent Antibody Technique / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009043:Motor Activity / Q000502:physiology; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D053495:Osteopontin; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D013119:Spinal Cord Injuries / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D019063:Tenascin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D012333:RNA, Messenger; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D019063:Tenascin; D053495:Osteopontin; D008668:Metallothionein; D002402:Cathepsin D