Introduction: Intervertebral disc (IVD) degeneration is often associated with low back pain and radiating leg pain. The purpose of this study is to develop a reproducible and standardized preclinical model of painful lumbar IVD degeneration by evaluation of structural and behavioral changes in response to IVD injury with increasing needle sizes. This model can be used to develop new therapies for IVD degeneration.Methods: Forty-five female Sprague Dawley rats underwent anterior lumbar disc needle puncture at levels L4-5 and L5-6 under fluoroscopic guidance. Animals were randomly assigned to four different experimental groups: needle sizes of 18 Gauge (G), 21G, 23G, and sham control. To monitor the progression of IVD degeneration and pain, the following methods were employed: μMRI, qRT-PCR, histology, and biobehavioral analysis.Results: T1-and T2-weighted μMRI analysis showed a correlation between the degree of IVD degeneration and needle diameter, with the most severe degeneration in the 18G group. mRNA expression of markers for IVD degeneration markers were dysregulated in the 18G and 21G groups, while pro-nociceptive markers were increased in the 18G group only. Hematoxylin and Eosin (H&E) and Alcian Blue/ Picrosirius Red staining confirmed the most pronounced IVD degeneration in the 18G group. Randall-Selitto and von Frey tests showed increased hindpaw sensitivity in the 18G group. Conclusion:Our findings demonstrate that anterior disc injury with an 18G needle creates severe IVD degeneration and mechanical hypersensitivity, while the 21G needle results in moderate degeneration with no increased pain sensitivity. Therefore, needle sizes should be selected depending on the desired phenotype for the pre-clinical model.

BackgroundIntervertebral disc (IVD) degeneration is a common cause of lower back pain, which carries substantial morbidity and economic cost. Omega-3 fatty acids (n-3 FA) are known to reduce inflammatory processes with a relatively benign side effect profile. This study aimed to investigate the effect of n-3 FA supplementation on IVD degeneration.Material/MethodsTwo non-contiguous lumbar discs of 12 Sprague Dawley rats were needle-punctured to induce disc degeneration. Post-surgery, rats were randomly assigned to either a daily n-3 FA diet (530 mg/kg/day of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in a 2: 1 ratio, administered in sucrose solution) or control diet (sucrose solution only), which was given for the duration of the study. After 1 month, blood serum arachidonic acid/eicosapentaenoic acid (AA/EPA) ratios were analyzed. After 2 months, micro-MRI (magnetic resonance imaging) analysis and histological staining of disc explants were performed to analyze the IVD.ResultsA reduction of blood AA/EPA ratios from 40 to 20 was demonstrated after 1 month of daily supplementation with n-3 FA. Micro-MRI analysis showed an injury-induced reduction of IVD hydration, which was attenuated in rats receiving n-3 FA. Histological evaluation demonstrated the destruction of nucleus pulposus tissue in response to needle puncture injury, which was less severe in the n-3 FA diet group.ConclusionsThe results of this study suggest that n-3 FA dietary supplementation reduces systemic inflammation by lowering AA/EPA ratios in blood serum and has potential protective effects on the progression of spinal disc degeneration, as demonstrated by reduced needle injury-induced dehydration of intervertebral discs and reduced histological signs of IVD degeneration.

Recombinant human bone morphogenic protein-2 (BMP-2)-loaded absorbable collagen sponges (ACS) have been successfully used to enhance bone formation and to induce spinal fusion in humans. However, side effects, such as soft tissue edema and inflammation, have been reported. NEMO binding domain peptide (NBD) inhibits activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a central regulator of immune response. In this study, we investigated NBD's potential to reduce BMP-2-induced soft tissue inflammation without affecting BMP-2-mediated spinal fusion in rat. For evaluation of soft tissue inflammation, ACS containing BMP-2, BMP-2+NBD, NBD, or ACS only were implanted into intramuscular paraspinal sites of 32 rats. At day 2 postsurgery, edema formation at the implant sites was assessed using magnetic resonance imaging. T2-weighted relaxation time (T2-RT) values were increased in the BMP-2 group compared with BMP-2+NBD, NBD, and ACS groups. No difference in T2-RT values was detected between BMP-2+NBD versus NBD and ACS controls. Postsacrifice, histological analysis of the implant-surrounding zones showed increased mononuclear cell infiltration in the BMP-2 group compared with BMP-2+NBD and controls. The presence of BMP-2 increased relative NF-κB binding and gene expression of inflammatory markers, interleukin (IL)1β, IL6, IL18, and chemokine ligand (CCL)2 and CCL3 compared with controls. In the BMP-2+NBD group, cytokine expression was blocked. No differences were found between BMP-2+NBD and control groups. For evaluation of spinal fusion, posterolateral intertransverse lumbar fusion procedures were performed on 16 rats. ACS were loaded with BMP-2 or BMP-2+NBD. After sacrifice at week 12, microcomputed tomographic assessment of the fusion site detected a higher bone volume and reduced trabecular spacing in the BMP-2+NBD group compared with BMP-2. Histological analysis did not show any differences in newly formed bone microarchitecture. In summary, addition of NBD to BMP-2-loaded ACS reduces BMP-2-induced soft tissue edema formation and mononuclear cell infiltration, diminishes NF-κB binding, and thus blocks transcription of NF-κB-regulated cytokines in rat. Furthermore, NBD stimulates bone formation in BMP-2-mediated spinal fusion, possibly through crosstalk of the NF-κB pathway with other pathways. The results of this study might provide the basis to develop new therapeutic bone grafting approaches with combinatory administration of BMP-2 and NBD for spinal fusion.

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