Withdrawn - Post Registration Deadline 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

Introduction: In 2022, 29.5 million Americans were diagnosed with alcohol use disorder (AUD), with less than 10% receiving treatment. AUD has been linked to chronic back pain and increased intervertebral disc degeneration (IDD)^1,2,3. One hallmark of IDD is cellular senescence which is known to induce catabolic shifts that are associated with inflammation and matrix degradation^4-6. Building on our previous observation that chronic alcohol consumption leads to early degenerative changes in the form of altered matrix morphology and stiffness, this study explores the mechanism behind alcohol-induced intervertebral disc degeneration⁷.

Methods: Mouse experiments were performed with IACUC approval. Adolescent (8-10 weeks-old) C57BL/6J mice were randomly divided into control (CTR; n=5/sex) and ethanol (EtOH; n=5 male, n=4 female) groups. To achieve a constant blood alcohol volume of 0.12%, EtOH mice were acclimated to ethanol in their drinking water over a period of 2 weeks, starting at 0% w/v ethanol and increasing 5% w/v every 3-4 days until reaching 20% w/v ethanol, which then was maintained for 12 weeks. Post-euthanasia, spines were analyzed for aggrecan degradation by immunohistochemistry in L3-5 region and Nucleus Pulposus (NP) protein was extracted from caudal IVDs to measure the level of NITEGE and NF-κB activity using western blot. Human NP cells (1 female, 2 male, Pfirrmann grade 1, obtained from autopsy donors) were cultured under 5% O₂. Cells from passage 3 were exposed to an Acetaldehyde Generating System (AGS, 0.02U/L ADH, 90mM NAD+, and 0.3% EtOH), which continually produces physiologically relevant levels of acetaldehyde, the main metabolite of EtOH. RNA was collected at 24, 48, 72, and 96 hours to assess P21 (cellular senescence), Aggrecan (matrix degradation), and CXCL2 (inflammation)expression. Data were analyzed using GraphPad prism. Unpaired t-tests with Welch’s correction were performed, and significance was set at p<0.05.

Results: Chronic ethanol (EtOH) consumption in mice significantly increased the activation of acetylated NF-κB and level of NITEGE expression (Figure 1A, B). Immunohistochemistry showed there is significant decrease of aggrecan levels in the IVD in male mice only (Figure 1C). In human NP cells, AGS exposure reduced Aggrecan expression, increased CXCL2 and p21 expression (Figure 2), and increase cell senescence, confirmed by elevated β-galactosidase staining (Figure 3A, B). 

Discussion: Chronic alcohol consumption accelerates intervertebral disc degeneration (IDD), although the specific mechanism by which alcohol contributes to IDD are not yet fully elucidated. The AGS system used in our in vitro experiment has been utilized in prior studies to produce acetaldehyde in cell cultures, where yeast alcohol dehydrogenase (ADH) metabolizes ethanol in the presence of NAD, simulating alcohol metabolism⁸. Our data demonstrated the harmful effect of chronic alcohol consumption on IVD health, affecting the nucleus pulposus (NP). There is a significant elevation in pro-inflammatory cytokines NF-κB and acetylated-NF-κB, as well as NITEGE in EtOH treated mice NP cells. In male mice, Aggrecan content was notably decreased, suggesting impaired extracellular matrix integrity. EtOH metabolism accelerates premature senescence (elevated P21 expression and beta galactosidase positive cells) in NP cells due to increase of inflammation (elevated CXCL2 expression) and matrix degradation (decreased Aggrecan expression).