Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro.
BACKGROUND
The application of mechanical insults to the spinal cord results in profound cellular and molecular changes, including the induction of neuronal cell death and altered gene expression profiles. Previous studies have described alterations in gene expression following spinal cord injury, but the specificity of this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile stresses on cultured spinal cord cells from E15 Sprague-Dawley rats, using the FX3000 Flexercell Strain Unit. We examined cell morphology and viability over a 72 hour time course. Microarray analysis of gene expression was performed using the Affymetrix GeneChip System, where categorization of identified genes was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. Changes in expression of 12 genes were validated with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).


RESULTS
The application of cyclic tensile stress reduced the viability of cultured spinal cord cells significantly in a dose- and time-dependent manner. Increasing either the strain or the strain rate independently was associated with significant decreases in spinal cord cell survival. There was no clear evidence of additive effects of strain level with strain rate. GO analysis identified 44 candidate genes which were significantly related to "apoptosis" and 17 genes related to "response to stimulus". KEGG analysis identified changes in the expression levels of 12 genes of the mitogen-activated protein kinase (MAPK) signaling pathway, which were confirmed to be upregulated by RT-PCR analysis.


CONCLUSIONS
We have demonstrated that spinal cord cells undergo cell death in response to cyclic tensile stresses, which were dose- and time-dependent. In addition, we have identified the up regulation of various genes, in particular of the MAPK pathway, which may be involved in this cellular response. These data may prove useful, as the accurate knowledge of neuronal gene expression in response to cyclic tensile stress will help in the development of molecular-based therapies for spinal cord injury.
Uchida
Kenzo
K
Department of Orthopaedics and Rehabilitation Medicine, Fukui University Faculty of Medical Sciences, Shimoaizuki 23, Matsuoka, Fukui 910-1193, Japan. kuchida@u-fukui.ac.jp
Nakajima
Hideaki
H
Hirai
Takayuki
T
Yayama
Takafumi
T
Chen
Ke-Bing
KB
Kobayashi
Shigeru
S
Roberts
Sally
S
Johnson
William E
WE
Baba
Hisatoshi
H
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002478:Cells, Cultured; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D020935:MAP Kinase Signaling System / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism*; D013314:Stress, Mechanical; D013312:Stress, Physiological / Q000235:genetics*; D013997:Time Factors; D015854:Up-Regulation / Q000235:genetics