Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

SOD2 Modulates Intervertebral Disc Degeneration during Aging (#204)

Shota Tamagawa 1 2 , Daisuke Sakai 1 , Hidetoshi Nojiri 2 , Takayuki Warita 1 3 , Jordy Schol 1 , Takahiko Shimizu 4 , Masato Sato 1 , Muneaki Ishijima 2 , Masahiko Watanabe 1
  1. Department of Orthopaedic Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan
  2. Department of Medicine for Orthopaedics and Motor Organ, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan
  3. TUNZ Pharma Inc., Osaka, Japan
  4. National Center for Geriatrics and Gerontology, Obu, Osaka, Japan

INTRODUCTION: Emerging evidence suggests that oxidative stress contributes to intervertebral disc (IVD) degeneration, but the precise underlying mechanisms remain elusive. The degree of oxidative stress is determined by the balance between reactive oxygen species (ROS) production and antioxidant enzyme activity. Superoxide dismutase 2 (SOD2) is a key antioxidant enzyme localized in mitochondria, a major site of ROS production. We have shown that the expression of SOD2 and oxidative stress markers in human IVD increases with the progression of disc degeneration and aging.1 We hypothesized that an imbalance between SOD2 and ROS accelerates disc degeneration. This study aimed to investigate whether Sod2 deficiency in IVD affects the progression of age-related disc degeneration in vivo.

 

METHODS: Lumbar and coccygeal IVDs of 6-, 12-, and 18-month-old (M) Sod2fl/fl (wild-type; WT) and chondrocyte-specific Sod2 conditional knockout (cKO) mice (Col2a1-Cre; SOD2fl/fl) were analyzed (n = 6 per group).2 First, we assessed mitochondrial ROS levels using MitoSOX fluorescence staining. Mean fluorescence intensity (MFI) in the nucleus pulposus (NP) and annulus fibrosus (AF) was quantified. Second, morphological changes were evaluated by hematoxylin/eosin and safranin-O/fast green staining. Histological scoring was performed using the ORS spine histopathological scoring system.3 Finally, we evaluated the expression of SOD2, p16INK4a, and type II collagen by immunohistochemistry. Statistical analysis was performed with 2-way ANOVA, followed by Tukey's post hoc test using GraphPad Prism.

 

RESULTS: MitoSOX MFI in NP was 1.31-fold higher in 12M WT mice and 1.69-fold higher in 18M WT mice (P = 0.0259) compared to 6M WT mice. MitoSOX MFI in AF was 1.56-fold higher in 12M WT mice (P = 0.0058) and 2.02-fold higher in 18M WT mice (P < 0.0001) compared to 6M WT mice. Furthermore, MitoSOX MFI was significantly higher in Sod2 cKO mice than in WT mice at all ages. Histological analysis revealed that Sod2 deficiency accelerated age-related disc degeneration in both lumbar and coccygeal IVDs. Specifically, WT mice showed only mild disc degeneration at 18M (average score; lumbar: 10.3, coccygeal: 9.6), whereas Sod2 cKO mice displayed significantly expedited disc degeneration at 18M (average score; lumbar: 17.1, coccygeal: 17.2). Immunohistochemical results confirmed the successful deletion of SOD2 in lumbar and coccygeal IVDs, as indicated by the complete loss of SOD2 positive cells in NP, AF and endplate regions. Additionally, Sod2 cKO mice showed significantly higher p16INK4a positivity and significantly lower type II collagen positivity than WT mice.

 

DISCUSSION: Previous studies have not elucidated whether oxidative stress accelerates disc degeneration or is merely a consequence of aging. Our study demonstrated that mitochondrial ROS in the IVD increases with aging and Sod2 deficiency accelerates disc degeneration through dysregulated ROS turnover, leading to cellular senescence and matrix degradation. The finding that elevated ROS in the IVD (observed in 12M WT mice) precedes histologically evident disc degeneration (observed in 18M WT mice) suggests that oxidative stress is a critical cause of disc degeneration. These results suggest that SOD2 plays a pivotal role in maintaining redox homeostasis in the IVD and mitochondrial ROS is a promising therapeutic target for IVD degeneration.

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  1. Tamagawa S, et al. Immunohistochemical Analysis of SOD2 Expression and Oxidative Stress Markers in Human Intervertebral Discs. ISSLS 2023.
  2. Koike M, et al. Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration. Sci Rep 2015.
  3. Melgoza IP, et al. Development of a standardized histopathology scoring system using machine learning algorithms for intervertebral disc degeneration in the mouse model-An ORS spine section initiative. JOR Spine 2021.