Longitudinal case-controlled animal study. To investigate putative cellular mechanisms to explain structural changes in muscle and adipose and connective tissues of the back muscles after intervertebral disc (IVD) injury. Structural back muscle changes are ubiquitous with back pain/injury and considered relevant for outcome, but their exact nature, time course, and cellular mechanisms remain elusive. We used an animal model that produces phenotypic back muscle changes after IVD injury to study these issues at the cellular/molecular level. Multifidus muscle was harvested from both sides of the spine at L1–L2 and L3–L4 IVDs in 27 castrated male sheep at 3 (n = 10) or 6 (n = 17) months after a surgical anterolateral IVD injury at both levels. Ten control sheep underwent no surgery (3 mo, n = 4; 6 mo, n = 6). Tissue was harvested at L4 for histological analysis of cross-sectional area of muscle and adipose and connective tissue (whole muscle), plus immunohistochemistry to identify proportion and cross-sectional area of individual muscle fiber types in the deepest fascicle. Quantitative polymerase chain reaction measured gene expression of typical cytokines/signaling molecules at L2. Contrary to predictions, there was no multifidus muscle atrophy (whole muscle or individual fiber). There was increased adipose and connective tissue (fibrotic proliferation) cross-sectional area and slow-to-fast muscle fiber transition at 6 but not 3 months. Within the multifidus muscle, increases in the expression of several cytokines (tumor necrosis factor α and interleukin-1β) and molecules that signal trophic/atrophic processes for the 3 tissue types (e.g., growth factor pathway [IGF-1, PI3k, Akt1, mTOR], potent tissue modifiers [calcineurin, PCG-1α, and myostatin]) were present. This study provides cellular evidence that refutes the presence of multifidus muscle atrophy accompanying IVD degeneration at this intermediate time point. Instead, adipose/connective tissue increased in parallel with the expression of the genes that provide putative mechanisms for multifidus structural remodeling. This provides novel targets for pharmacological and physical interventions. Level of Evidence: N/A

Study Design. Longitudinal case-controlled animal study. Objective. To investigate the effect of an intervertebral disc (IVD) lesion on the proportion of slow, fast, and intermediate muscle fiber types in the multifidus muscle in sheep, and whether muscle fiber changes were paralleled by local gene expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1-β. Summary of Background Data. Structure and behavior of the multifidus muscle change in acute and chronic back pain, but the mechanisms are surprisingly poorly understood and the link between structure and behavior is tenuous. Although changes in muscle fiber types have the potential to unify the observations, the effect of injury on muscle fiber distribution has not been adequately tested, and understanding of possible mechanisms is limited. Methods. The L1–L2, L3–L4, and L5–L6 IVDs of 11 castrated male sheep received anterolateral lesions. Six control sheep underwent no surgical procedures. Multifidus muscle tissue was harvested at L4 for muscle fiber analysis using immunohistochemistry and L2 for cytokine analysis with polymerase chain reaction for local gene expression of TNF-α and interleukin-1β. Results. The proportion of slow muscle fibers in multifidus was significantly less in the lesioned animals both ipsilateral and contralateral to the IVD lesion. The greatest reduction in slow fibers was in the deep medial muscle region. A greater prevalence of intermediate fibers on the uninjured side implies a delayed fiber-type transformation on that side. TNF-α gene expression in multifidus was greater on both sides in the lesion animals than in the muscle of control animals. Interleukin-1β was increased only on the injured side. Conclusion. These data provide evidence of muscle fiber changes after induction of an IVD lesion and a parallel increase in TNF-α expression. Proinflammatory cytokine changes provide a novel mechanism to explain behavioral and structural changes in multifidus. Level of Evidence: N/A

An investigation of mechanical destabilization of the lumbar ovine intervertebral disc (IVD) inducing IVD degeneration (IVDD) as determined by multiparameter outcome measures (magnetic resonance imaging [MRI], IVD composition, biomechanical testing, gene profiling, immunohistochemistry, and immunoblotting). To assess the effect of IVD mechanical destabilization on matrix protein and metalloproteinase gene expression to investigate the pathophysiological mechanisms of lumbar IVDD. Several earlier studies have used annular transection to induce IVDD in sheep, but none have optimized or validated the most appropriate lesion size. The annulus fibrosus (AF) incision inducing maximal change in IVD biomechanics was applied to L1–L2, L3–L4, and L5–L6 discs in vivo to compare with a sham procedure at 3 months post operation. IVDs were evaluated by MRI, biomechanics, histopathology, proteoglycan and collagen content, gene expression, and aggrecan proteolysis by Western blotting. Significant changes were observed in lesion (6 × 20 mm2) compared with sham IVDs at 3 months post operation: reduced disc height on MRI; increased neutral zone in biomechanical testing; depleted proteoglycan and collagen content in the nucleus pulposus (NP) and lesion half of the AF but not in the contralateral AF; increased messenger RNA for collagen I and II, aggrecan, versican, perlecan, matrix metalloproteinase (MMP)-1 & 13, and ADAMTS-5, in the lesion-site AF and NP but not in the contralateral AF. ADAMTS-4 messenger RNA was increased in the lesion-site AF but decreased in the NP. Despite an upregulation in MMPs, there was no change in MMP- or ADAMTS-generated aggrecan neoepitopes in any region of the IVD in lesion or sham discs. Lumbar IVDD was reproducibly induced with a 6 × 20 mm2 annular lesion, with focal dysregulation of MMP gene expression, cell cloning in the inner AF, loss of NP aggrecan, and disc height. Loss of aggrecan from the NP was not attributable to increased proteolysis in the interglobular domain by MMPs or ADAMTS.

Objective: To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG‐2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene‐knockout model.Methods: Maturational changes were evaluated histologically in the knees of 3‐, 6‐, and 12‐week‐old wild‐type (WT) mice and Hspg2^{Δ3−/Δ3−} mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2^{Δ3−/Δ3−} mice. Changes in cartilage expression of FGF‐2, FGF‐18, HSPG‐2, FGF receptor 1 (FGFR‐1), and FGFR‐3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin‐1α (IL‐1α), FGF‐2, and FGF‐18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified.Results: No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2^{Δ3−/Δ3−} mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA‐induced loss of chondrocyte expression of FGF‐2, FGF‐18, and HSPG‐2 occurred in both genotypes. Expression of FGFR‐1 after OA induction was maintained in WT mice, while FGFR‐3 loss after OA induction was significantly reduced in Hspg2^{Δ3−/Δ3−} mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL‐1α–stimulated cartilage. However, IL‐1α–induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2^{Δ3−/Δ3−} mice. Cartilage GAG release in either the presence or absence of IL‐1α was unaltered by FGF‐2 in both genotypes. In cartilage cultures with FGF‐18, IL‐1α–stimulated GAG loss was significantly reduced only in Hspg2^{Δ3−/Δ3−} mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA.Conclusion: Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR‐3 and increased FGF signaling.