Longitudinal changes in the structure and inflammatory response of the intervertebral disc due to stab injury in a murine organ culture model

Abstract

Despite the significant public health impact of intervertebral disc (IVD) degeneration and low back pain, it remains challenging to investigate the multifactorial molecular mechanisms that drive the degenerative cascade. Organ culture model systems offer the advantage of allowing cells to live and interact with their native extracellular matrix, while simultaneously reducing the amount of biological variation and complexity present at the organismal level. Murine organ cultures in particular also allow the use of widely available genetically modified animals with molecular level reporters that would reveal insights on the degenerative cascade. Here, we utilize an organ culture system of murine lumbar functional spinal units where we are able to maintain the cellular, metabolic, and structural, and mechanical stability of the whole organ over a 21-day period. Furthermore, we describe a novel approach in organ culture by using tissues from animals with an NF-κB-luc reporter in combination with a mechanical injury model, and are able to show that proinflammatory factors and cytokines such as NF-κB and IL-6 produced by IVD cells can be monitored longitudinally during culture in a stab injury model. Taken together, we utilize a murine organ culture system that maintains the cellular and tissue level behavior of the intervertebral disc and apply it to transgenic animals that allow the monitoring of the inflammatory profile of IVDs. This approach could provide important insights on the molecular and metabolic mediators that regulate the homeostasis of the IVD.

An <em>In Vitro</em> Organ Culture Model of the Murine Intervertebral Disc

“…With aging and degeneration, the IVD components change structurally, compositionally, and mechanically 4 . These changes include the loss of proteoglycans and hydration in the NP, decreased disc height, and deteriorated mechanical competence 5 . These alterations are often accompanied by cytokines that promote an inflammatory response, as well as neutrophil and dorsal root ganglion intrusion into the joint space culminating in a cascade of events that lead to LBP symptoms 6 .…”

“…Thus, a reductionist approach of simplifying the experimental system down to the IVD organ allows mechanistic fine-tuning of causal variables and examining their downstream effects 5 . The system is reduced to only the native cell population and surrounding extracellular matrix, thus enabling the direct interpretation of the effects of external stimuli on IVD degeneration.…”

An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate

“…Recently, a stab injury model, based on motion segments isolated from transgenic animals with an NF-κB-luc reporter, was developed. Longitudinal monitoring of NF-κB activity in this model can provide insights into the mechanisms that regulate IVD inflammatory responses to stab injury [46]. At the same time, physiologically relevant cytokine-induced inflammatory organ culture models, without involvement of mechanical damage, have emerged [7,47,48].…”

The Quantitative Structural and Compositional Analyses of Degenerating Intervertebral Discs Using Magnetic Resonance Imaging and Contrast-Enhanced Micro-Computed Tomography

“…By design, this study does not account for the cellular level effects that may occur with trypsin or the stab, although both are shown to produce structural and inflammatory changes 19–21,33,34 . Further, the microCT-derived intensity attenuation values of the NP require regional normalization to either the AF in order to compute meaningful values of NI/DI.…”

“…To determine the ability of this technique to monitor IVD degeneration, we propose to apply this approach to monitor the cascade in several established IVD degeneration models: puncturing of the IVD, injection of trypsin, and injection of collagenase. The puncture of the IVD using a needle mimics a mechanical injury by physically breaching the IVD 18,19 . Trypsin is a serine protease that catalyzes the hydrolysis of peptide bonds, and previous studies showed that trypsin injection into the NP deteriorates IVD mechanical behavior and GAGs content 20,21 .…”