Abstract
BackgroundThe 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).ResultsThe 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.ConclusionsWe 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.
Highlights
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
Transmission Electron Microscopy (TEM) examination showed that all cells at 0 hours appeared viable, with large nuclei, and dotted with chromatin and an abundant rough endoplasmic reticulum
The cell survival rate in cultures subjected to a 15% strain level at a frequency of 0.5 Hz was significantly lower than that seen at the standard level
Summary
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. Mechanical stresses applied to the spinal cord can potentially induce profound and irreversible paresis, secondary to induced pathological changes such as dysfunction and loss of neurons, impairment of neuronal cell survival mechanisms and protein synthesis, neuronal cell necrosis and apoptosis [1,2] Examples of such mechanically induced spinal cord damage include spinal cord compression but distraction insult [3,4]; it is Changes in the expression of several immediate early response genes have been documented in various in vivo models of spinal cord injury using microarray analysis [911]. The differential and post-traumatic expression levels of several genes have been explored in vivo in an attempt to stabilize, both biologically and functionally, the spinal cord once injured [12,13] These in vivo experimental settings for studying the response of neuronal systems to mechanical injury suffer from several disadvantages over in vitro experimentation. In vitro models of the spinal cord stimuli can be useful to gain a better understanding of the specific neuronal response to mechanical stress
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