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An organ culture system to model early degenerative changes of the intervertebral disc II: profiling global gene expression changes.

ABSTRACT: Despite many advances in our understanding of the molecular basis of disc degeneration, there remains a paucity of preclinical models which can be used to study the biochemical and molecular events that drive disc degeneration, and the effects of potential therapeutic interventions. The goal of this study is to characterize global gene expression changes in a disc organ culture system that mimics early nontraumatic disc degeneration.To mimic a degenerative insult, rat intervertebral discs were cultured in the presence of TNF-?, IL-1? and serum-limiting conditions. Gene expression analysis was performed using a microarray to identify differential gene expression between experimental and control groups. Differential pattern of gene expression was confirmed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or Western blot.Treatment resulted in significant changes in expression of more than 1,000 genes affecting many aspects of cell function including cellular movement, the cell cycle, cellular development, and cell death and proliferation. Many of the most highly upregulated and downregulated genes have known functions in disc degeneration and extracellular matrix hemostasis. Construction of gene networks based on known cellular pathways and expression data from our analysis demonstrated that the network associated with cell death, cell cycle regulation and DNA replication and repair was most heavily affected in this model of disc degeneration.This rat organ culture model uses cytokine exposure to induce wide gene expression changes with the most affected genes having known reported functions in disc degeneration. We propose that this model is a valuable tool to study the etiology of disc degeneration and evaluate potential therapeutic treatments.

SUBMITTER: Markova DZ

PROVIDER: S-EPMC3978582 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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An organ culture system to model early degenerative changes of the intervertebral disc II: profiling global gene expression changes.

Markova Dessislava Z DZ Kepler Christopher K CK Addya Sankar S Murray Hallie B HB Vaccaro Alexander R AR Shapiro Irving M IM Anderson D Greg DG Albert Todd J TJ Risbud Makarand V MV

Arthritis research & therapy 20130101 5

<h4>Introduction</h4>Despite many advances in our understanding of the molecular basis of disc degeneration, there remains a paucity of preclinical models which can be used to study the biochemical and molecular events that drive disc degeneration, and the effects of potential therapeutic interventions. The goal of this study is to characterize global gene expression changes in a disc organ culture system that mimics early nontraumatic disc degeneration.<h4>Methods</h4>To mimic a degenerative in ...[more]

PMID: 24171898

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Project description:IntroductionThe goal of this study is to characterize transcriptome changes and gene regulation networks in an organ culture system that mimics early post-traumatic intervertebral disc (IVD) degeneration.MethodsTo mimic a traumatic insult, bovine caudal IVDs underwent one strike loading. The control group was cultured under physiological loading. At 24 hours after one strike or physiological loading, RNA was extracted from nucleus pulposus (NP) and annulus fibrosus (AF) tissue. High throughput next generation RNA sequencing was performed to identify differentially expressed genes (DEGs) between the one strike loading group and the control group. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze DEGs and pathways. Protein-protein interaction (PPI) network was analyzed with cytoscape software. DEGs were verified using qRT-PCR. Degenerated human IVD tissue was collected for immunofluorescence staining to verify the expression of DEGs in human disc tissue.ResultsOne strike loading resulted in significant gene expression changes compared with physiological loading. In total 253 DEGs were found in NP tissue and 208 DEGs in AF tissue. Many of the highly dysregulated genes have known functions in disc degeneration and extracellular matrix (ECM) homeostasis. ACTB, ACTG, PFN1, MYL12B in NP tissue and FGF1, SPP1 in AF tissue were verified by qRT-PCR and immunofluorescence imaging. The identified DEGs were involved in focal adhesion, ECM-receptor interaction, PI3K-AKT, and cytokine-cytokine receptor interaction pathways. Three clusters of PPI networks were identified. GO enrichment revealed that these DEGs were mainly involved in inflammatory response, the ECM and growth factor signaling and protein folding biological process.ConclusionOur study revealed different DEGs, pathways, biological process and PPI networks involved in post-traumatic IVD degeneration. These findings will advance the understanding of the pathogenesis of IVD degeneration, and help to identify novel biomarkers for the disease diagnosis.

Project description:BackgroundAcute trauma on intervertebral discs (IVDs) is thought to be one of the risk factors for IVD degeneration. The pathophysiology of IVD degeneration induced by single high impact mechanical injury is not very well understood. The aim of this study was using a post-traumatic IVD model in a whole organ culture system to analyze the biological and biomechanical consequences of the single high-impact loading event on the cultured IVDs.MethodsIsolated healthy bovine IVDs were loaded with a physiological loading protocol in the control group or with injurious loading (compression at 50% of IVD height) in the one strike loading (OSL) group. After another 1 day (short term) or 8 days (long term) of whole organ culture within a bioreactor, the samples were collected to analyze the cell viability, histological morphology and gene expression. The conditioned medium was collected daily to analyze the release of glycosaminoglycan (GAG) and nitric oxide (NO).ResultsThe OSL IVD injury group showed signs of early degeneration including reduction of dynamic compressive stiffness, annulus fibrosus (AF) fissures and extracellular matrix degradation. Compared to the control group, the OSL model group showed more severe cell death (P < 0.01) and higher GAG release in the culture medium (P < 0.05). The MMP and ADAMTS families were up-regulated in both nucleus pulposus (NP) and AF tissues from the OSL model group (P < 0.05). The OSL injury model induced a traumatic degenerative cascade in the whole organ cultured IVD.ConclusionsThe present study shows a single hyperphysiological mechanical compression applied to healthy bovine IVDs caused significant drop of cell viability, altered the mRNA expression in the IVD, and increased ECM degradation. The OSL IVD model could provide new insights into the mechanism of mechanical injury induced early IVD degeneration.The translational potential of this articleThis model has a high potential for investigation of the degeneration mechanism in post-traumatic IVD disease, identification of novel biomarkers and therapeutic targets, as well as screening of treatment therapies.

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An organ culture system to model early degenerative changes of the intervertebral disc II: profiling global gene expression changes.

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An organ culture system to model early degenerative changes of the intervertebral disc II: profiling global gene expression changes.

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