In situ oxygen utilization in the rat intervertebral disc

Abstract

Nucleus pulposus cells of the intervertebral disc have no endogenous vasculature and have thus been hypothesized to be hypoxic. This hypothesis was tested using 2-nitroimidazole, EF5, a drug that at low oxygen concentrations forms covalent adducts with cellular proteins. After administrating EF5 to rats, sections of the intervertebral disc were analysed for EF5 adducts. Drug adducts were quantified in tissue sections using a fluorescent monoclonal antibody. Although the level of EF5 fluorescence in all intervertebral disc tissues was low, the transition zone at the periphery of the nucleus pulposus exhibited the highest level of EF5 binding. To substantiate this result, tissue nitroreductase levels and drug pharmacology were evaluated. Nitroreductase levels were measured in whole discs under severe hypoxia. We noted that there was robust EF5 binding to cells in the annulus fibrosus and transition zone with modest binding to cells of the nucleus pulposus and endplate. High-performance liquid chromatography analysis indicated limitations in EF5 access to the nucleus pulposus, most probably related to the lack of vasculature and slow drug distribution through the gel-like interior of the disc. However, despite diffusion problems, the drug dose was determined to be sufficient to report the oxygen status of the nucleus pulposus cells. Based on these findings, we conclude that despite poor vascularization, the disc cells accommodate to the local environment by displaying a limited need for oxygen. Accordingly, the cells of the intervertebral disc are not severely hypoxic.

ROS: Crucial Intermediators in the Pathogenesis of Intervertebral Disc Degeneration

“…In other words, ROS production is a price paid for the aerobic metabolism. While the microenvironment of IVDs is characterized by hypoxia due to poor vascularization, all resident disc cells (NP cells, AF cells, and CEP cells) have been demonstrated to be not anaerobic and to have oxygen-utilizing metabolic processes in vivo [49, 50]. Therefore, disc cells are expected to produce ROS in the microenvironment of discs.…”

Tonicity enhancer binding protein (TonEBP) and hypoxia-inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: Role of Hsp70 in HIF-1α degradation

“…It consists of an outer fibrocartilagenous annulus fibrosus enclosing a gel-like nucleus pulposus that contains cells embedded in an abundant aggrecan rich extracellular matrix that imparts tissue its osmotic properties. A limited number of blood vessels infiltrate the outer one third of the annulus fibrosus but in no case enter the nucleus pulposus (1–5). In this hypoxic and hypertonic microenvironment, nucleus pulposus cells express several key transcription factors including HIF-1α and its homologue HIF-2α (6, 7), regulators of glycolytic metabolism (8) and oxidative activities (9, 10) respectively and Tonicity enhancer binding protein (TonEBP), an important osmoregulator (11).…”

Environmental regulation of notochordal gene expression in nucleus pulposus cells

“…Finally, because the disc is avascular, nutrient supply-particularly, oxygen-has been thought to be a strong determinant of IVD form and function during growth (Urban et al, 2004;Grunhagen et al, 2006). Aside from MAPK signaling and VEGF expression changes in rat NP cells (Risbud et al, 2005a,b;Agrawal et al, 2008;Fujita et al, 2008), hypoxia does not appear to generate oxaemic distress (Rajpurohit et al, 2002;Risbud et al, 2006;Lee et al, 2007). However, it is not clear how hypoxia might regulate other genes associated with the immature NP such as CD24, type IIA collagen, a 6 integrin subunit, and galectin-3 (Zhu et al, 2001;Hunter et al, 2003;Fujita et al, 2005;Chen et al, 2006;Gilchrist et al, 2007;Zeng et al, 2007).…”