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IntroductionIntervertebral disc degeneration is a leading cause of chronic low back pain (LBP), but how degeneration contributes to LBP is poorly understood. Nerve growth factor (NGF) levels increa...

Introduction Increasing evidence suggests that healthy and painless intervertebral discs (IVDs) are largely aneural and that degenerating and painful IVDs often are innervated.1 Many inflammatory factors are unregulated in disc degeneration, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), and are believed to play important roles in degeneration and pain.2,3 Increased levels of neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), have also been associated with disc degeneration and could potentiate disc innervation.4 Inflammatory factors and neurotrophins can also have nociceptive roles that promote the development of chronic pain. While increased levels of inflammatory and nociceptive factors have been found by histological analysis in degenerating discs, in disc cell cultures, and in animal models,3 the ability of degenerating intervertebral discs from chronic low back pain patients to release increased levels of nociceptive factors compared with healthy discs from pain free donors is unknown. An understanding of the ability of degenerating discs to release nociceptive factors that may sensitize neurons and the mechanisms involved will provide evidence to develop effective pain management strategies. Materials and Methods Degenerating, painful IVDs were collected from patients undergoing surgery for low back pain and healthy, painless IVDs were collected from human organ donors through Transplant Quebec. IVDs were cultured ex vivo for 48 hours and the conditioned media was collected. Protein arrays were used to assess relative quantities of 23 cytokines and chemokines and enzyme-linked immunosorbent assay (ELISA) kits were used to quantify TNF-α, NGF, and BDNF in the conditioned media. PC12 cells, a neuronal-like cell line, were exposed to the conditioned or NGF supplemented media and neurite sprouting was analyzed. Mouse sensory neurons were isolated from dorsal root ganglia and treated with conditioned media. Neuronal expression of CGRP, a peptide involved in pain modulation, was compared with the total number of neurons that were stained with PGP 9.5, a general neuronal marker and the percentage of CGRP immunoreactive neurons was determined. To determine the role of NGF in neurite sprouting and CGRP regulation NGF supplemented or degenerating IVD conditioned media was incubated with an anti-NGF antibody. Cytokine array data were analyzed by unpaired t-tests and neurite sprouting and CGRP expression was assessed by one-way ANOVA. Results Cytokine arrays found that degenerating painful IVDs released significantly higher levels of a majority of the factors represented in the array as compared with healthy painless IVDs. Many of them are nociceptive factors including IFN-γ, IL-6, MCP-1, GRO-α. IELISA assays demonstrated higher levels of the NGF and BDNF, in media from degenerating painful discs. Increased neurite growth was observed in PC12 cell cultures maintained in degenerating IVD media compared with healthy IVD media ( p < 0.001). Anti-NGF antibodies were able to prevent neurite growth to the level found in healthy IVD media cultures ( p = 0.929). In addition, a greater number of the mouse neurons cultured in degenerating IVD conditioned media were CGRP immunoreactive compared with those cultured in healthy IVD media ( p = 0.007). The number of CGRP immunoreactive neurons was significantly reduced in the presence of anti-NGF antibodies ( p > 0.999). Conclusion Increased levels of pronociceptive and neurotrophic factors released by degenerating painful IVDs could potentiate IVD innervation and neuronal hypersensitization. Prolonged neuronal exposure to these factors could promote the development of chronic pain. The induced neurite growth and CGRP expression suggests that degenerating IVDs secrete factors that can lead to IVD innervation and nociception. NGF inhibition studies found NGF released by degenerating IVDs is required for the increased neurite growth and CGRP expression, providing evidence to support an important role for NGF in chronic low back pain associated with disc degeneration. The roles of factors besides NGF in disc degeneration require further investigation. These results suggest that degenerating, painful IVDs produce factors, which may cause neoinnervation and pain in vivo. Furthermore, this study provides evidence to support further development of anti-NGF therapies as a pain management strategy for low back pain associated with disc degeneration. Disclosure of Interest None declared References Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O’Brien J, Jayson MI. Nerve ingrowth into diseased intervertebral disc in chronic back pain. Lancet 1997;350(9072):178–181 Le Maitre CL, Freemont AJ, Hoyland JA. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration. Arthritis Res Ther 2005;7(4):R732-R745 Wuertz K, Haglund L. Inflammatory mediators in intervertebral disk degeneration and discogenic pain. Global Spine J 2013;3(3):175–184 Richardson SM, Purmessur D, Baird P, Probyn B, Freemont AJ, Hoyland JA. Degenerate human nucleus pulposus cells promote neurite outgrowth in neural cells. PLoS ONE 2012;7(10):e47735

Introduction Chronic low back pain (LBP) affects over 10% of the adult population resulting in profound decreases in the quality of life. The intervertebral disk (IVD) is prone to degeneration which may contribute toLBP. To prevent disk degeneration-related LBP, better understanding of disk structure and the degenerative process is needed to understand the underlying mechanisms. Standard Hematoxylin and Eosin (H&E) staining, alone or in combination with other dyes, have been used to assess the normal biology and degeneration of IVDs. Recently, the multichromatic FAST (Alcian blue, Safranin-O, Fast green and Tartrazine) staining method has been developed (Leung V.Y.L. et al., 2009). This method provides additional information unavailable through conventional methods such as differentiation of the structures within the disk and identification of degenerative matrix remodeling through changes in the staining pattern. Although the FAST method has proved helpful in animal models to assess alteration of glycosaminoglycan content, its usefulness to assess human disk histopathology remains unknown. Materials and Methods Human disks were obtained (a) surgically from chronic LBP patients with moderate-severe disk degeneration or (b) postmortem from transplant donors. Disks were cut into quadrants, fixed in 4% paraformaldehyde with 14% (v/v) saturated picric acid for 3 days, embedded in OCT and stored at −80°C. Tissue samples were cut on a cryostat and transverse sections of 14 um and 20 um were obtained for H&E and FAST staining, respectively. Slides were coverslipped with DPX and scanned using a Zeiss MIRAX Scan Digital Slide Scanner for further examination. Results The FAST protocol creates high-resolution and polychromatic images of the general disk structure, and reveals the overall organization of the disk. The transition from the nucleus pulposus (NP) to the inner annulus fibrosus (IAF) and the outer annulus fibrosus (OAF) was clearly visible through color boundaries. Signs of degeneration could be observed with the FAST staining method in both IVDs obtained postmortem with no history of LBP and from chronic LBP patients. For example, FAST staining uncovered the loss of a clear boundary between the NP and the IAF that was not visible with H&E. These data suggest that relevant pathological details might be missed if assessing histological changes with the use of H&E only. Conclusion The combined use of H&E and FAST methods provides a more complete picture of the anatomy and physiology of human IVDs. The FAST profile distinguishes IVD compartments and shows matrix remodeling events within the disk, while H&E provides complementary information regarding the cellular content of the tissue. These two staining methods complement each other to give a clearer differentiation of IVD sub-regions of both normal and degenerated disk architecture. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Leung V.Y.L. et al. Journal of Histochemistry and Cytochemistry 2009;57:249

Introduction Chronic low back pain (LBP) affects over 10% of the adult population and seriously decreases quality of life. The aging intervertebral disk (IVD) is prone to degeneration, which is associated with an increased risk of LBP. (Luoma et al., 2000) Physical activity influences IVD physiology. Animal studies show that cyclic loading increases proteoglycan synthesis in the disk suggesting a role for running exercise in disk repair. (Brisby et al., 2010) Additionally, an active lifestyle has been shown to either contribute to the onset of chronic pain onset or to be protective against chronic pain depending on the circumstances. Our laboratory has validated a rodent model of LBP, the aging SPARC-null mouse. These mice demonstrate accelerated lumbar disk degeneration as they age, similar to the degeneration observed in human subjects with LBP. SPARC-null mice also display axial and radiating pain that mirrors LBP in humans and is pharmacologically reversible. (Millecamps, Tajerian, Sage, & Stone, 2011) Hypothesis Habitual running exercise will delay disk degeneration and decrease pain behavior in aging SPARC-null mice. Materials and Methods Total 2- to 3-month-old male SPARC-null and C57B wild type mice were used in this study. After recording baseline behavioral measurements, animals had free access to either a circular treadmill (exercise group) or a fixed nonrotating treadmill (control group) in their home cages. Axial LBP was assessed using the tail suspension test and radiating LBP was assessed using the acetone test for cold hypersensitivity in the hindpaw. SPARC-null mice exhibit signs of both axial and radiating LBP in these assays that is sensitive to pharmacological manipulation. Additional assays included tests for mechanical sensory thresholds and motor capacity. Tests were performed at 2 weeks and monthly thereafter. Results After 2 months with access to running wheels, SPARC-null mice show significantly reduced cold sensitivity in the hindpaw compared to baseline ( p Conclusion Total 2 months of voluntary running exercise partially reversed the development of radiating LBP in SPARC-null mice. Reversal of axial pain may require longer exposure. Future studies These mice will be followed for 6 months postwheel exposure. Following sacrifice, disk degeneration will be assessed using radiographic and histological methods. Disk innervation will be determined by immunohistochemistry, and the relationships between anatomical and behavioral measures will be determined. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Brisby, H., Wei, A. Q., Molloy, T., Chung, S. A., Murrell, G. A., Diwan, A. D. The effect of running exercise on intervertebral disk extracellular matrix production in a rat model Spine 2010;35(15):1429–1436 Luoma, K., Riihimäki, H., Luukkonen, R., Raininko, R., Viikari-Juntura, E., Lamminen, A. Low back pain in relation to lumbar disk degeneration Spine 2000;25(4):487–492 Millecamps, M., Tajerian, M., Sage, E. H., Stone, L. S. Behavioral signs of chronic back pain in the SPARC-null mouse Spine 2011;36(2):95–102