Background Intervertebral disc degeneration contributes to low back pain. The avascular intervertebral disc consists of a central hypoxic nucleus pulpous (NP) surrounded by the more oxygenated annulus fibrosus (AF). Lactic acid, an abundant end-product of NP glycolysis, has long been viewed as a harmful waste that acidifies disc tissue and decreases cell viability and function. As lactic acid is readily converted into lactate in disc tissue, the objective of this study was to determine whether lactate could be used by AF cells as a carbon source rather than being removed from disc tissue as a waste byproduct. Methods Import and conversion of lactate to tricarboxylic acid (TCA) cycle intermediates and amino acids in rabbit AF cells were measured by heavy-isotope (13C-lactate) tracing experiments using mass spectrometry. Levels of protein expression of lactate converting enzymes, lactate importer and exporter in NP and AF tissues were quantified by Western blots. Effects of lactate on proteoglycan (35S-sulfate) and collagen (3H-proline) matrix protein synthesis and oxidative phosphorylation (Seahorse XFe96 Extracellular Flux Analyzer) in AF cells were assessed. Results Heavy-isotope tracing experiments revealed that AF cells imported and converted lactate into TCA cycle intermediates and amino acids using in vitro cell culture and in vivo models. Addition of exogenous lactate (4 mM) in culture media induced expression of the lactate importer MCT1 and increased oxygen consumption rate by 50%, mitochondrial ATP-linked respiration by 30%, and collagen synthesis by 50% in AF cell cultures grown under physiologic oxygen (2-5% O2) and glucose concentration (1-5 mM). AF tissue highly expresses MCT1, LDH-H, an enzyme that preferentially converts lactate to pyruvate, and PDH, an enzyme that converts pyruvate to acetyl-coA. In contrast, NP tissue highly expresses MCT4, a lactate exporter, and LDH-M, an enzyme that preferentially converts pyruvate to lactate. Conclusions These findings support disc lactate-dependent metabolic symbiosis in which lactate produced by the hypoxic, glycolytic NP cells is utilized by the more oxygenated AF cells via oxidative phosphorylation for energy and matrix production, thus shifting the current research paradigm of viewing disc lactate as a waste product to considering it as an important biofuel. These scientifically impactful results suggest novel therapeutic targets in disc metabolism and degeneration.
Objective: Rabbit annulus fibrosus (AF) cells were exposed to isolated or combined mechanical and inflammatory stress to examine the expression of neuropeptide Y (NPY). This study aims to explore the ability of AF cells to produce NPY in response to mechanical and inflammatory stress. Methods: Lumbar AF cells of 6-to 8-month-old female New Zealand white rabbits were harvested and exposed to combinations of inflammatory (interleukin-1β) and mechanical (6% or 18%) tensile stress using the Flexcell System. NPY concentrations were measured in the media via enzyme-linked immunosorbent assay. The presence of NPY receptor-type 1 (NPY-1R) in AF cells of rabbit intervertebral discs was also analyzed via immunohistochemistry and immunofluorescence. Results: Exposure to inflammatory stimuli showed a significant increase in the amount of NPY expression compared to control AF cells. Mechanical strain alone did not result in a significant difference in NPY expression. While combined inflammatory and mechanical stress did not demonstrate an increase in NPY expression at low (6%) levels of strain, at 18% strain, there was a large-though not statistically significant-increase in NPY expression under conditions of inflammatory stress. Lastly, immunofluorescence and immunohistochemistry of AF cells and tissue, respectively, demonstrated the presence of NPY-1R. Conclusion: These findings demonstrate that rabbit AF cells are capable of expressing NPY, and expression is enhanced in response to inflammatory and mechanical stress. Because both inflammatory and mechanical stress contribute to intervertebral disc degeneration (IDD), this observation raises the potential of a mechanistic link between low back pain and IDD.
Background: Previous animal models of intervertebral disc degeneration (IDD) rely on open surgical approaches, which confound the degenerative response and pain behaviors due to injury to surrounding tissues during the surgical approach. To overcome these challenges, we developed a minimally invasive percutaneous puncture procedure to induce IDD in a rat model.Methods: Ten Fischer 344 male rats underwent percutaneous annular puncture of lumbar intervertebral discs (IVDs) at L2-3, L3-4, and L4-5. Ten unpunctured rats were used as controls. Magnetic resonance imagings (MRIs), serum biomarkers, and behavioral tests were performed at baseline and 6, 12, and 18 weeks post puncture. Rats were sacrificed at 18 weeks and disc histology, immunohistochemistry, and glycosaminoglycan (GAG) assays were performed.Results: Punctured IVDs exhibited significant reductions in MRI signal intensity and disc volume. Disc histology, immunohistochemistry, and GAG assay results were consistent with features of IDD. IVD-punctured rats demonstrated significant changes in pain-related behaviors, including total distance moved, twitching frequency, and rearing duration.Conclusions: This is the first reported study of the successful establishment of a reproducible rodent model of a percutaneous lumbar annular puncture resulting in discogenic pain. This model will be useful to test therapeutics and elucidate the basic mechanisms of IDD and discogenic pain.
Objective. The aim of this study was to determine the effect of graft type on residual motion and the relationship among residual motion, smoking, and patient-reported outcome (PRO) scores following anterior cervical discectomy and fusion (ACDF). Summary of Background Data. Although most patients develop solid fusion based on static imaging following ACDF, dynamic imaging has revealed that many patients continue to have residual motion at the arthrodesis. Methods. Forty-eight participants performed dynamic neck flexion/extension and axial rotation within a biplane radiography system 1 year following ACDF (21 one-level, 27 two-level). PRO scores included the Short Form-36, Neck Disability Index, and Cervical Spine Outcomes Questionnaire. An automated modelbased tracking process matched subject-specific bone models to the biplane radiographs with sub-millimeter accuracy. Residual motion was measured across the entire arthrodesis site for both one-and two-level fusions in patients who received either allograft or autograft. Patients were divided into ''pseudarthrosis'' (>38 of flexion/extension residual motion) and ''solid fusion'' groups. Residual motion and PROs were compared between groups using Student t tests.
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