Dataset Information

Modulation of the In Vivo Inflammatory Response by Pro- Versus Anti-Inflammatory Intervertebral Disc Treatments.

ABSTRACT: Inflammation is central in intervertebral disc (IVD) degeneration/regeneration mechanisms, and its balance is crucial to maintain tissue homeostasis. This work investigates the modulation of local and systemic inflammatory response associated with IVD degeneration/herniation by administration of PRO- versus ANTI-inflammatory treatments. Chitosan/poly-?-glutamic acid nanocomplexes, known as pro-inflammatory (PRO), and soluble diclofenac, a non-steroidal anti-inflammatory drug (ANTI), were intradiscally administered in a rat IVD injury model, 24 h after lesion. Two weeks after administration, a reduction of disc height accompanied by hernia formation was observed. In the PRO-inflammatory treated group, IL-1?, IL-6 and COX-2 IVD gene expression were upregulated, and loss of nucleus pulposus (NP) structure and composition was observed. Systemically, lower T-cell frequency was observed in the lymph nodes (LN) and spleen (SP) of the PRO group, together with an increase in CD4+ T cells subset in the blood (BL) and LN. In contrast, the ANTI-group had higher proteoglycans/collagen ratio and collagen type 2 content in the NP, while an increase in the frequency of myeloid cells, M1 macrophages and activated macrophages (MHCII+) was observed at the systemic level. Overall, this study illustrates the dynamics of local and systemic inflammatory and immune cell responses associated with intradiscal therapies, which will contribute to designing more successful immunomodulatory treatments for IVD degeneration.

SUBMITTER: Cunha C

PROVIDER: S-EPMC7084831 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Modulation of the In Vivo Inflammatory Response by Pro- Versus Anti-Inflammatory Intervertebral Disc Treatments.

Cunha Carla C Q Teixeira Graciosa G Ribeiro-Machado Cláudia C L Pereira Catarina C Ferreira Joana R JR Molinos Maria M G Santos Susana S Barbosa Mário A MA M Goncalves Raquel R

International journal of molecular sciences 20200303 5

Inflammation is central in intervertebral disc (IVD) degeneration/regeneration mechanisms, and its balance is crucial to maintain tissue homeostasis. This work investigates the modulation of local and systemic inflammatory response associated with IVD degeneration/herniation by administration of PRO- versus ANTI-inflammatory treatments. Chitosan/poly-γ-glutamic acid nanocomplexes, known as pro-inflammatory (PRO), and soluble diclofenac, a non-steroidal anti-inflammatory drug (ANTI), were intradi ...[more]

PMID: 32138314

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Project description:Discogenic low back pain (LBP) is a main cause of disability and inflammation is presumed to be a major driver of symptomatic intervertebral disc degeneration (IDD). Anti-inflammatory agents are currently under investigation as they demonstrated to alleviate symptoms in patients having IDD. However, their underlying anti-inflammatory and regenerative activity is poorly explored. The present study sought to investigate the potential of Etanercept and Tofacitinib for maintaining disc homeostasis in a preclinical intervertebral disc (IVD) organ culture model within IVD bioreactors allowing for dynamic loading and nutrient exchange. Bovine caudal IVDs were cultured in a bioreactor system for 4 days to simulate physiological or degenerative conditions: (1) Phy-physiological loading (0.02-0.2 MPa; 0.2 Hz; 2 h/day) and high glucose DMEM medium (4.5 g/L); (2) Deg+Tumor necrosis factor ? (TNF-?)-degenerative loading (0.32-0.5 MPa; 5 Hz; 2 h/day) and low glucose DMEM medium (2 g/L), with TNF-? injection. Etanercept was injected intradiscally while Tofacitinib was supplemented into the culture medium. Gene expression in the IVD tissue was measured by RT-qPCR. Release of nitric oxide (NO), interleukin 8 (IL-8) and glycosaminoglycan (GAG) into the IVD conditioned medium were analyzed. Cell viability in the IVD was assessed using lactate dehydrogenase and ethidium homodimer-1 staining. Immunohistochemistry was performed to assess protein expression of IL-1?, IL-6, IL-8, and collagen type II in the IVD tissue. Etanercept and Tofacitinib downregulated the expression of IL-1?, IL-6, IL-8, Matrix metalloproteinase 1 (MMP1), and MMP3 in the nucleus pulposus (NP) tissue and IL-1?, MMP3, Cyclooxygenase-2 (COX2), and Nerve growth factor (NGF) in the annulus fibrosus (AF) tissue. Furthermore, Etanercept significantly reduced the IL-1? positively stained cells in the outer AF and NP regions. Tofacitinib significantly reduced IL-1? and IL-8 positively stained cells in the inner AF region. Both, Etanercept and Tofacitinib reduced the GAG loss to the level under physiological culture condition. Etanercept and Tofacitinib are able to neutralize the proinflammatory and catabolic environment in the IDD organ culture model. However, combined anti-inflammatory and anabolic treatment may be required to constrain accelerated IDD and relieving inflammation-induced back pain.

Project description:Low back pain is a highly prevalent clinical problem and intervertebral disc (IVD) degeneration is now accepted as the major pathophysiological mechanism responsible for this condition. Accumulating evidence suggests that inflammation plays a crucial role in the progression of human IVD degeneration, with macrophages being pointed as the key immune cell players in this process since their infiltration in degenerated IVD samples has been extensively demonstrated. Since they are highly plastic, macrophages can play different roles depending on the microenvironmental cues. The study of inflammation associated with IVD degeneration has been somehow neglected and one of the reasons is related with lack of adequate models. To overcome this, we established and characterized a new model of IVD organ culture under pro-inflammatory conditions to further dissect the role of macrophages in IVD associated immune response. For that, human monocyte-derived macrophages were co-cultured either with bovine caudal IVD punches in the presence of the pro-inflammatory cytokine IL-1?, or IVD-conditioned medium (CM), to investigate how IVD-produced factors influence macrophage phenotype. After 72 h, metabolic activity, gene expression and cytokine profile of macrophages and IVD cells were measured. Our results show that macrophages and IVDs remain metabolically active in the presence of IL-1?, significantly upregulate CCR7 gene expression and increase production of IL-6 on macrophages. When treating macrophages with IL-1?-IVD-CM, CCR7 upregulation follows the same trend, while for IL-6 an opposite effect was observed. On the other hand, macrophages interfere with IVD ECM remodeling, decreasing MMP3 expression and downregulating aggrecan and collagen II gene expression in the presence of IL-1?. Overall, the co-culture model established in this study can be considered a suitable approach to address the cellular and molecular pathways that regulate macrophage-IVD crosstalk, suggesting that degenerated IVD tissue tends to polarize human macrophages toward a more pro-inflammatory profile, which seems to aggravate IVD degeneration. This model could be used to improve the knowledge of the mechanisms that link IVD degeneration and the immune response.

Project description:Intervertebral disc herniation (IVDH) is an important pathology in humans and also in dogs. While the molecular disease mechanisms are well investigated in humans, little is known about the inflammatory mediators in naturally occurring canine IVDH. The objective of this study was to investigate whether the involved proinflammatory cytokines in human IVDH are also key cytokines in canine IVDH and thus to elucidate the suitability of the dog as a model for human trials. 59 samples from 25 dogs with surgically confirmed thoracolumbar IVDH were collected and classified in three subgroups: herniated (H), affected non-herniated (NH) disc, and adjacent non-affected (NA) disc. Discs from 11 healthy dogs acted as controls (C). Samples were analyzed for IL-1, IL-6, IL-8, and TNF-? expression (qPCR/ELISA) as well as cell infiltration and activation of the MAP kinase pathways (immunohistochemistry). Gene and protein expression of all key cytokines could be detected in IVDH affected dogs. Canine IVDH was significantly associated with a higher gene expression of IL-6 (H?>?C, NH?>?C) and TNF-? (H?>?C, NH?>?C, NA?>?C) and a significant down-regulation of IL-1? (H?<?C). Dogs with spontaneous pain had significantly higher IL-6 mRNA compared to those with pain arising only upon palpation. An inter-donor comparison (H and HN relative to NA) revealed a significant increase of IL-6 gene expression (H?>?NA, NH?>?NA). IL-8 (H?>?C, NA?>?C) and TNF-? (NH?>?C) protein levels were significantly increased in diseased dogs while inversely, IL-6 protein levels were significantly higher in patients with better clinical outcome. Aside from resident IVD cells, mostly monocytes and macrophages were found in extruded material, with concomitant activation of extracellular signal-regulated kinase p38 in the majority of samples. Dogs with spontaneous IVDH might provide a useful model for human disc diseases. Although the expression of key cytokines found in human IVDH was also demonstrated in canine tissue, the inflammatory mechanisms accompanying canine IVDH diverges partially from humans, which will require further investigations in the future. In dogs, IL-6 seems to play an important pathological role and may represent a new potential therapeutic target for canine patients.

Project description:BackgroundSpinal growth modulation has been proposed as a non-fusion strategy for treatment of idiopathic scoliosis, although the effect of this treatment modality on intervertebral disc health has not been evaluated in detail. The objectives of this in vivo study were to assess the creation of three-dimensional spinal deformity during six months of growth modulation compared with that in sham-surgery controls, and to compare, with use of magnetic resonance imaging (MRI), gross morphological, histological, and biochemical analyses, disc health between control animals and animals treated with a spinal tether.MethodsSix immature Yucatan mini-pigs underwent anterior spinal instrumentation with vertebral screws connected by a polyethylene tether over four consecutive thoracic vertebrae (T8-T11). An additional six animals underwent sham surgery (screw placement only [the control group]). Radiographs were obtained preoperatively, postoperatively, and monthly thereafter during six months of growth. Computed tomography (CT) and MRI studies were performed ex vivo, and the spines were sectioned for histological and biochemical analyses. Multivariate analysis of variance (MANOVA) was used to compare six-month postoperative data between the control and tethered animals, with the alpha level of significance set at 0.05.ResultsRadiographs and CT images demonstrated the creation of significant three-dimensional deformity (p < 0.013) in the tethered animals compared with the controls. Macroscopic, MRI, and histological evaluation revealed no signs of disc degeneration, with a bulging gelatinous nucleus pulposus, discrete fibrous anular lamellae, and uniformly hyperintense T2-signal intensity within the nuclei pulposi. Biochemical analysis demonstrated no significant difference in the nuclei pulposus between the tethered and control vertebrae; however, the water content (p < 0.001) of both sides of the anulus fibrosus and the glycosaminoglycan content (p < 0.001) of the left side of the anulus fibrosus differed significantly between the two groups.ConclusionsSix months of spinal growth modulation created significant spinal deformity in all three planes compared with what was found in the sham-surgery controls. Although disc health was qualitatively maintained, quantitative changes in the anulus fibrosus water content and the disc height were observed on the side opposite to the tether. These changes likely represent metabolic responses of the discs to compressive loads generated by the flexible tether.

Dataset Information

Modulation of the In Vivo Inflammatory Response by Pro- Versus Anti-Inflammatory Intervertebral Disc Treatments.

Publications

Modulation of the In Vivo Inflammatory Response by Pro- Versus Anti-Inflammatory Intervertebral Disc Treatments.

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