Takashi Yurube

Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration

IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

Gene therapy approach for disc degeneration and associated spinal disorders

Disc degeneration is deeply associated with many spinal disorders and thus has a significant clinical impact on society. The currently available surgical treatment often necessitates removing a pathological disc and spinal fusion. However, it is also well known that these surgical treatments have many potential problems including invasion and cost. Therefore, biological approaches for regenerating these pathological discs have received much attention. Gene therapy is one of these biological approaches. Gene therapy involves the transfer of genes to cells so the recipient cells express these genes and thereby synthesize the RNA and protein they encode in a continuous fashion. One of the significant advantages of gene therapy is that we can expect a lasting duration of biological effect which is potentially beneficial for most disc degeneration associated disorders, as they are, by nature, chronic conditions. Originally, gene therapy was mediated by viral vectors, but recent technological progress has enabled us to opt for non-virus-mediated gene therapy for the disc. Furthermore, the development of the RNA interference technique has enabled us to down-regulate a specific gene expression in the disc opening the door for a new generation of intradiscal gene therapy.

Progression of Cervical Spine Instabilities in Rheumatoid Arthritis : A Prospective Cohort Study of Outpatients over 5 Years

Study Design. A 5-year prospective cohort study of cervical spine instabilities in rheumatoid arthritis (RA). Objective. To clarify the natural course of cervical instabilities in RA patients and to determine predictors for the prognosis of RA cervical spine. Summary of Background Data. Although several previous studies investigating the natural history of RA cervical spine have been reported, few of them have described radiological predictive factors for the aggravation of these instabilities. Methods. Two hundred sixty-seven outpatients with “definite” or “classical” RA initially assigned were prospectively followed for over 5 years. Radiographic cervical findings were classified into three representative instabilities: atlantoaxial subluxation (AAS), vertical subluxation (VS), and subaxial subluxation (SAS). The aggravations of these instabilities were identified in the cases with a decrease of at least 2 mm in the Ranawat value of VS, an increase of at least 1 mm in translation of SAS, or a new development of respective instabilities. RA stages and mutilating changes were assessed in the hand radiograms. Results. Fifty-two point four percent of 267 patients, without any cervical instability at the beginning of follow-up, decreased to 29.6% at the end (P < 0.01), whereas VS and SAS increased significantly (P < 0.01). The aggravation of VS was observed at statistically higher rates in patients with pre-existing instabilities as follows; 25.7% of AAS (P = 0.01), 49.1% of VS (P < 0.01), and 41.2% of SAS (P = 0.06). The aggravation of SAS was also detected in 47.2% of VS and 64.7% of SAS (P < 0.01). Patients with pre-existing mutilating changes exhibited the aggravations of VS and SAS in significantly higher incidences (P < 0.01). Furthermore, the cases with development into mutilating changes during the follow-up showed significantly higher tendencies for the aggravations of these instabilities (P < 0.01). Conclusion. The incidences of VS and SAS significantly increased during the minimum 5-year follow-up. Prognostic factors of these instabilities were revealed to be the initial radiological findings of VS, SAS, and mutilating changes.

Fas ligand plays an important role for the production of pro‐inflammatory cytokines in intervertebral disc nucleus pulposus cells

It is suggested that pro‐inflammatory cytokines, which are produced by interaction of the intervertebral nucleus pulposus cells and macrophages, may be linked to the cause of pain of the intervertebral disc herniation. This study carries out the in vitro experiments to examine the mechanism, with the use of the co‐culture of an immortalized cell line of nucleus pulposus of the human intervertebral disc and the macrophage cell line. As a result, it is found that the production of pro‐inflammatory cytokines is significantly larger at the co‐culture group than at the independent culture group. Also, at the co‐culture group of macrophages and intervertebral nucleus pulposus cells with over‐expression of fas ligand (FasL), the production of pro‐inflammatory cytokines is found to be far larger. Furthermore, it is found that these pro‐inflammatory cytokines are produced mainly by the intervertebral nucleus pulposus cells with over‐expression of FasL, and that the expression of a disintegrin and metalloproteinase (ADAM) 10, which controls the expression of FasL and activates reverse signaling inside cells, also increases. From these findings, it is suggested that FasL and ADAM10 play an important role in the production of pro‐inflammatory cytokines coming from interaction of the intervertebral nucleus pulposus cells and macrophages.

Intervertebral disc and macrophage interaction induces mechanical hyperalgesia and cytokine production in a herniated disc model in rats

OBJECTIVE The expression of proinflammatory factors such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), IL-8, and prostaglandin E(2) (PGE(2) ) is significantly correlated with the symptoms of herniated disc disease. Among the different types of immune cells, macrophages are frequently noted in the herniated disc tissue. We undertook this study to clarify the interaction of the intervertebral disc (IVD) and macrophages with regard to the production of TNFα, IL-6, IL-8, and PGE(2) . METHODS We developed 2 animal models to assess the interactions of IVDs with macrophages in terms of TNFα, IL-6, IL-8, and PGE(2) production and pain-related behavior. We also cocultured IVDs and macrophages to assess the role of TNFα in IL-6, IL-8, and PGE(2) production. RESULTS IVD autografts induced TNFα, IL-6, IL-8, and cyclooxygenase 2 (COX-2) messenger RNA (mRNA) up-regulation; macrophage infiltration was seen shortly after the autograft was implanted. A significant decrease was noted in the mechanical threshold of the ipsilateral paw following the up-regulation of TNFα, IL-6, IL-8, and COX-2 mRNA. Only IVD and macrophage cocultures resulted in IL-8 and PGE(2) up-regulation. TNFα up-regulation was maximized before that of IL-6 and IL-8. TNFα neutralization attenuated production of IL-6 and PGE(2) , but not that of IL-8. Neutralization of TNFα and IL-8 significantly increased the paw withdrawal mechanical threshold in the IVD autograft and spinal nerve ligation model. CONCLUSION IVD-macrophage interaction plays a major role in sciatica and in the production of TNFα, IL-6, IL-8, and PGE(2) . TNFα is required for IL-6 and PGE(2) production, but not for IL-8 production, during IVD-macrophage interaction. Neutralization of TNFα and IL-8 can be a valuable therapy for herniated disc disease.

Incidence and aggravation of cervical spine instabilities in rheumatoid arthritis: a prospective minimum 5-year follow-up study of patients initially without cervical involvement.

Study Design. A prospective minimum 5-year follow-up study of the cervical spine in patients with rheumatoid arthritis (RA) initially without cervical involvement. Objective. To clarify the incidence and aggravation of cervical spine instabilities and their predictive risk factors in patients with RA. Summary of Background Data. Many reports have shown the progression of cervical spine involvement in RA. However, few articles have described comprehensive evaluation of its prognostic factors. Methods. A total of 140 patients with “definite” or “classical” RA initially without cervical involvement were prospectively followed for more than 5 years. Radiographical cervical findings were classified into 3 instabilities: atlantoaxial subluxation (AAS: atlantodental interval >3 mm), vertical subluxation (VS: Ranawat value <13 mm), and subaxial subluxation (SAS: irreducible translation ≥2 mm). “Severe” extents were defined as AAS with atlantodental interval 10 mm or more, VS with Ranawat value 10 mm or less, and SAS with translation 4 mm or more or at multiple levels. Incidence of these developments and predictors for “severe” instabilities were investigated. Results. During 6.0 ± 0.5 years, 43.6% of 140 patients developed cervical instabilities: AAS in 32.1%, VS in 11.4%, and SAS in 16.4% with some combinations. “Severe” instabilities were exhibited in 12.9% of patients: AAS in 3.6%, VS in 6.4%, and SAS in 5.0%. Furthermore, 4.3% presented canal stenosis, with 13 mm or less space available for the spinal cord (SAC) due to “severe” AAS or “severe” VS in 2.9% and 12 mm or less SAC due to “severe” SAS in 2.1%. Multivariable logistic regression analysis identified corticosteroid administration, mutilating changes at baseline, and the development of nonmutilating into mutilating changes during the follow-up period correlated with “severe” instabilities (P < 0.05). Conclusion. A minimum 5-year follow-up reveals the occurrence of cervical instabilities in 43.6%, “severe” aggravation in 12.9%, and decreased SAC in 4.3% of patients with RA. Characteristics of severe disease activity—established mutilating changes, progressive development into mutilating changes, and potentially concomitant corticosteroid treatment—are indicators for poor prognosis of the cervical spine in RA.

Matrix metalloproteinase (MMP)-3 gene up-regulation in a rat tail compression loading-induced disc degeneration model.

The rodent static compression loading‐induced disc degeneration model still has important gaps among the radiographic, magnetic resonance imaging (MRI), and histological schemes and the acute and chronic expression of catabolic genes such as matrix metalloproteinase (MMP)‐3. Our objectives were to assess the validity of a rat tail two‐disc static compression model and to elucidate a representative catabolic marker, MMP‐3 gene alterations, throughout the degenerative process. Static compression at 1.3 MPa for up to 56 days produced progressive disc height loss in radiographs, lower nucleus intensity on T2‐weighted MRIs, and histomorphological degeneration. Real‐time RT‐PCR mRNA quantification showed significant MMP‐3 up‐regulation in nucleus pulposus cells from 7 days and a significantly progressive increase as the loading duration lengthened, with high correlations to radiological degenerative scores. Immunohistochemistry demonstrated progressively increased positive staining for MMP‐3. These results validate this animal model for disc degeneration research. Progressive mRNA and protein‐distributional up‐regulations indicate the significant role of MMP‐3 and its feasibility as a disc degenerative marker. This model should prove useful for investigating the pathomechanism and for evaluating molecular therapies for degenerative disc disease.

A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype

The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types—notochordal cells (NCs) and non‐notochordal chondrocyte‐like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI‐positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin‐8‐ and galectin‐3‐co‐positive cells, identified as NCs. The proportion of TUNEL‐positive cells, which predominantly comprised non‐NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up‐regulation of ADAMTS‐5 in the 1‐day loaded group and MMP‐3 in the 7‐day loaded group. Aggrecan‐1 and collagen type 2α‐1 mRNA levels were down‐regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration.

Accelerated Development of Cervical Spine Instabilities in Rheumatoid Arthritis: A Prospective Minimum 5-Year Cohort Study

Objective To clarify the incidence and predictive risk factors of cervical spine instabilities which may induce compression myelopathy in patients with rheumatoid arthritis (RA). Methods Three types of cervical spine instability were radiographically categorized into “moderate” and “severe” based on atlantoaxial subluxation (AAS: atlantodental interval >3 mm versus ≥10 mm), vertical subluxation (VS: Ranawat value <13 mm versus ≤10 mm), and subaxial subluxation (SAS: irreducible translation ≥2 mm versus ≥4 mm or at multiple). 228 “definite” or “classical” RA patients (140 without instability and 88 with “moderate” instability) were prospectively followed for >5 years. The endpoint incidence of “severe” instabilities and predictors for “severe” instability were determined. Results Patients with baseline “moderate” instability, including all sub-groups (AAS+ [VS− SAS−], VS+ [SAS− AAS±], and SAS+ [AAS± VS±]), developed “severe” instabilities more frequently (33.3% with AAS+, 75.0% with VS+, and 42.9% with SAS+) than those initially without instability (12.9%; p<0.003, p<0.003, and p = 0.061, respectively). The incidence of cervical canal stenosis and/or basilar invagination was also higher in patients with initial instability (17.5% with AAS+, 37.5% with VS+, and 14.3% with SAS+) than in those without instability (7.1%; p = 0.028, p<0.003, and p = 0.427, respectively). Multivariable logistic regression analysis identified corticosteroid administration, Steinbrocker stage III or IV at baseline, mutilating changes at baseline, and the development of mutilans during the follow-up period correlated with the progression to “severe” instability (p<0.05). Conclusions This prospective cohort study demonstrates accelerated development of cervical spine involvement in RA patients with pre-existing instability—especially VS. Advanced peripheral erosiveness and concomitant corticosteroid treatment are indicators for poor prognosis of the cervical spine in RA.

Modified house-keeping gene expression in a rat tail compression loading-induced disc degeneration model.

House‐keeping genes (HKGs) are generally used as endogenous controls for molecular normalization in quantitative PCR analysis. However, whether all the so‐called HKGs are useful for intervertebral disc research is controversial. Our objective was, using a prevalidated rat tail static compression loading‐induced disc degeneration model, to clarify the feasibility of common HKGs for gene‐quantification in the nucleus pulposus cells. In real‐time RT‐PCR for five HKGs [β‐actin, β‐glucuronidase, β‐2 microglobulin, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA)], static compression at 1.3 MPa for up to 56 days demonstrated messenger RNA (mRNA) expression levels of consistent β‐2 microglobulin and GAPDH, slightly up‐regulated β‐glucuronidase, and fairly down‐regulated β‐actin and LDHA. Especially, β‐actin had a drastic suppression to 0.15‐fold in the loaded relative to unloaded discs at 7 days. In immunofluorescence, β‐actin showed a significant down‐regulation to almost undetectable levels from 28 days, while GAPDH was constantly detected throughout. β‐Actin mRNA and protein‐distribution are thought to be affected by the loading treatment, however, GAPDH mRNA and protein‐distribution can retain relatively stable expressions. Under prolonged static compression, β‐actin and probably LDHA are inappropriate, and GAPDH is a feasible HKG as internal references in the disc cells.