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The role of oxidative stress in ligamentum flavum (LF) hypertrophy has not been elucidated. We hypothesize that oxidative stress induces inflammatory responses and the subsequent fibrotic processes in LF, via activation of the Akt and MAPK pathways. Specimens of LFs were collected during surgeries for lumbar disc herniation (LDH) or lumbar spinal stenosis (LSS). Part of the LF specimens underwent analyses for ROS, fibrotic markers, and inflammatory mediators, with the remainder minced for cell cultures. The cell cultures were treated with H 2 O 2 , after which the cells were lysed and analyzed via western blotting. The specimens of the LSS patients showed increased infiltration of inflammatory cells and were stained positively for MMP-3, MMP-9, vimentin, and fibronectin. The LF of the LSS patients had increased oxidative stress and inflammation compared to that of the LDH patients. In vitro analyses demonstrated that oxidative stress rapidly activated the Akt and MAPK pathways. Inflammatory mediators, iNOS and NF-κB, and fibrotic markers, including TGF-β, β-catenin, α-SMA and vimentin, were significantly upregulated after induction of oxidative stress. Oxidative stress activated the intrinsic apoptotic pathway. These findings revealed that oxidative stress is one of the etiological factors of LF hypertrophy, which might provide new insights into treatment approaches.

Study Design: Animal experiment: a rat model of lumbar disc herniation (LDH) induced painful radiculopathies., Objective: To investigate the role and mechanism of AMP-activated protein kinase (AMPK) in dorsal root ganglia (DRG) neurons in LDH-induced painful radiculopathies., Summary of Background Data: Overactivation of multiple pain signals in DRG neurons triggered by LDH is crucial to the development of radicular pain. AMPK is recognized as a cellular energy sensor, as well as a pain sensation modulator, but its function in LDH-induced pain hypersensitivity remains largely unknown., Methods: The LDH rat model was established by autologous nucleus pulposus transplantation into the right lumbar 5 (L5) nerve root. At different time points after AMPK agonist metformin (250 mg/kg/d) or mammalian target of rapamycin (mTOR) inhibitor rapamycin (5 mg/kg) intraperitoneal administration, thermal and mechanical sensitivity were evaluated by measuring paw withdrawal latency (PWL) and 50% paw withdrawal thresholds (PWT). The levels of AMPK, mTOR, and p70S6K phosphorylation were determined by Western blot. We also investigated the proportion of p-AMPK positive neurons in the right L5 DRG neurons using immunofluorescence., Results: LDH evoked persistent thermal hyperalgesia and mechanical allodynia on the ipsilateral paw, as indicated by the decreased PWL and 50% PWT. These pain hypersensitive behaviors were accompanied with significant inhibition of AMPK and activation of mTOR in the associated DRG neurons. Pharmacological activation of AMPK in the DRG neurons not only suppressed mTOR/p70S6K signaling, but also alleviated LDH-induced pain hypersensitive behaviors., Conclusion: We provide a molecular mechanism for the activation of pain signals based on AMPK-mTOR axis, as well as an intervention strategy by targeting AMPK-mTOR axis in LDH-induced painful radiculopathies., Level of Evidence: N/A.

Background: This is an immunohistologic study of gene expression between patients and controls.This study aims to evaluate expression of the catalase gene in hypertrophied ligamentum flavum (LF) specimens obtained from patients with lumbar spinal canal stenosis (LSCS).LSCS is one of the most common spinal disorders. It is well known that LF hypertrophy plays an important role in the onset of LSCS. Although degenerative changes, aging, and mechanical stress are all thought to contribute to hypertrophy and fibrosis of the LF, the precise pathogenesis of LF hypertrophy remains unknown. Previous genetic studies have tried to determine the mechanism of LF hypertrophy. However, the association between catalase gene expression and LF hypertrophy has not yet been explored., Methods: LF specimens were surgically obtained from 30 patients with spinal stenosis (LSCS group) and from 30 controls with lumbar disc herniation (LDH group). LF thickness was measured at the thickest point using calipers to an accuracy of 0.01 mm during surgical intervention. The extent of LF elastin degradation and fibrosis were graded (grades 0-4) by hematoxylin and eosin staining and Masson trichrome staining, respectively. The resulting LF measurements, histologic data, and immunohistologic results were then compared between the 2 groups., Results: The average LF thickness was significantly higher in the LSCS group than in the LDH group (5.99 and 2.95 mm, respectively, P = .004). Elastin degradation and fibrosis of the LF were significantly more severe in spinal stenosis samples than in the disc herniation samples (3.04 ± 0.50 vs 0.79 ± 0.60, P = .007; 3.01 ± 0.47 vs 0.66 ± 0.42, P = .009, respectively). Significantly lower expression of catalase was observed in the perivascular area of LF samples obtained from patients with LSCS compared with controls (61.80 ± 31.10 vs 152.80 ± 41.13, respectively, P = .009)., Conclusion: Our findings suggest that decreased expression of catalase is associated with LF hypertrophy in patients with LSCS.

Aldehyde dehydrogenase (ALDH) is a key enzyme for the catalytic oxidation of acetaldehyde to acetic acid. Genetic polymorphisms of ALDH2 have been associated with a wide range of diseases and cancers. However, little information is found about the association between ALDH2 polymorphisms and lumbar disc herniation (LDH) in Chinese Han population. We investigated the association between single nucleotide polymorphisms (SNPs) in ALDH2 and LDH risk in a case-control study that included 380 LDH cases and 692 healthy controls. Eight SNPs were selected and genotyped using the Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression after adjusting for gender and age. In the allele model analysis, we found the frequency of the "A" allele of rs671 was significantly higher in LDH cases than in controls (OR = 1.414, 95%CI: 1.109-1.803, P = 0.005). In the genetic model analysis, we found the minor allele "A" of rs671 was associated with increased risk of LDH under log-additive model (OR = 1.42, 95%CI: 1.11-1.82, P = 0.0062); and the minor allele "C" of rs7296651 was associated with decreased risk of LDH under over-dominant model (OR = 0.72, 95%CI: 0.53-0.97, P = 0.031). Additionally, the haplotype "GGCTCACG" constructed by rs886205, rs2238152, rs4648328, rs441, rs4646778, rs671, rs11066028, and rs7296651 was associated with increased risk of LDH (OR = 1.45; 95% CI = 1.11-1.90; P = 0.0071). Our data shed new light on the association between genetic polymorphisms of ALDH2 and LDH susceptibility in a Chinese Han population.

Intervertebral disc (IVD) degeneration is responsible for various spine pathologies and present clinical treatments are insufficient. Concurrently, the mechanisms behind IVD degeneration are still not completely understood, so as to allow development of efficient tissue engineering approaches. A model of rat IVD degeneration directly coupled to herniation is here proposed in a pilot study. Disc injury is induced by needle puncture, using two different needles gauges: a low caliber 25-G needle and a high caliber 21-G needle. Histological, biochemical, and radiographic degeneration was evaluated at 2 and 6 weeks post-injury. We show that the larger caliber needle results in a more extended histological and radiographic degeneration within the IVD, compared to the smaller one. TUNEL quantification indicates also increased cell death in the 21-G group. Analyses of collagen type I (Picrosirius red staining), collagen type II (immunofluorescence), and GAG content (Blyscan assay) indicate that degeneration features spontaneously recover from 2 to 6 weeks, for both needle types. Moreover, we show the occurrence of hernia proportional to the needle gauge. The number of CD68+ macrophages present, as well as cell apoptosis within the herniated tissue are both proportional to hernia volume. Moreover, hernias formed after lesion tend to spontaneously diminish in volume after 6 weeks. Finally, MMP3 is increased in the hernia in the 21-G group at 2 weeks. This model, by uniquely combining IVD degeneration and IVD herniation in the same animal, may help to understand mechanisms behind IVD pathophysiology, such as hernia formation and spontaneous regression. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:258-268, 2017., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background Lumbar disc herniation is a major cause of radicular pain, but the underlying mechanisms remain largely unknown. Spinal activation of src-family kinases are involved in the development of chronic pain from nerve injury, inflammation, and cancer. In the present study, the role of src-family kinases activation in lumbar disc herniation-induced radicular pain was investigated. Results Lumbar disc herniation was induced by implantation of autologous nucleus pulposus, harvest from tail, in lumbar 4/5 spinal nerve roots of rat. Behavior test and electrophysiologic data showed that nucleus pulposus implantation induced persistent mechanical allodynia and thermal hyperalgesia and increased efficiency of synaptic transmission in spinal dorsal horn which underlies central sensitization of pain sensation. Western blotting and immunohistochemistry staining revealed that the expression of phosphorylated src-family kinases was upregulated mainly in spinal microglia of rats with nucleus pulposus. Intrathecal delivery of src-family kinases inhibitor PP2 alleviated pain behaviors, decreased efficiency of spinal synaptic transmission, and reduced phosphorylated src-family kinases expression. Furthermore, we found that the expression of ionized calcium-binding adapter molecule 1 (marker of microglia), tumor necrosis factor-α, interleukin 1 -β in spinal dorsal horn was increased in rats with nucleus pulposus. Therapeutic effect of PP2 may be related to its capacity in reducing the expression of these factors. Conclusions These findings suggested that central sensitization was involved in radicular pain from lumbar disc herniation; src-family kinases-mediated inflammatory response may be responsible for central sensitization and chronic pain after lumbar disc herniation.

To investigate Caspase-3 gene polymorphisms (rs4647693 G/A, rs4647610 A/G, and rs12108497 T/C) and susceptibility to lumbar intervertebral disc herniation (LDH). The genotype frequency distributions of the polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in 107 LDH patients (case group) and 121 healthy individuals (control group). SHEsis software was used to conduct gene linkage disequilibrium and haplotype analysis. Regression analysis was used to analyze possible risk factors for LDH. Statistically significant differences in family history of LDH, amateur sports, leisure activities, bed types, and spine load grade were found between the case and control groups. The distribution of allele and genotype frequencies of rs4647693 G/A, rs4647610 A/G, and rs12108497 T/C polymorphisms of Caspase-3 were significantly different between the case and control groups. Haplotype analysis showed that the G-G-C (rs4647693-rs4647610-rs12108497) haplotype might be a risk factor for LDH, whereas the A-A-T haplotype might be a protective factor (p < 0.05). Binary logistic regression analysis showed that the GA+AA genotype of rs4647693 was negatively associated with the risk of LDH, whereas high spine load grade was positively associated with the risk of LDH. These findings revealed that rs4647693 G/A, rs4647610 A/G, and rs12108497 T/C polymorphisms of Caspase-3 may be associated with susceptibility to LDH and that interaction and modification effects may exist between Caspase-3 polymorphisms., (Copyright © 2016 Kaohsiung Medical University. Published by Elsevier Taiwan.. All rights reserved.)

The present study was performed to investigate the role of p38 mitogen‑activated protein kinase (MAPK) in the resorption of herniated intervertebral discs in 30 rats. In the non‑contained and p38 MAPK inhibition (p38i) groups, two coccygeal intervertebral discs (IVDs) were removed and wounded prior to relocation into the subcutaneous space of the skin of the back. In the contained group, the cartilage endplates maintained their integrity. Furthermore, SB203580 was injected intraperitoneally into the p38i group, whereas saline was injected into the other two groups. In the non‑contained group, the weight of the relocated IVDs decreased to a greater extent over time when compared with the contained and p38i groups. Phosphorylated p38, tumor necrosis factor‑α, and interleukin‑1β were observed to exhibit higher expression levels in the non‑contained group compared with the contained and p38i groups, at weeks 1 and 4 post‑surgery. The expression level of caspase‑3 and the densities of apoptotic disc cells were significantly higher in the non‑contained group compared with the contained and p38i groups at 4 weeks post‑surgery. In conclusion, p38 MAPK induces apoptosis in IVDs, while also accelerating the resorption of the relocated IVDs. Thus, p38 MAPK may be important in spontaneous resorption of IVDs.

Aim: The intervertebral disc starts to degenerate when a human being begins to stand and learn to walk. It is known that many extrinsic, intrinsic and genetic factors play a role in disc degeneration. In this study, we examined whether the matrix metalloproteinase 11 might be associated with intervertebral disc degeneration., Material and Methods: Fifty-six patients with lumbar disc herniations who were operated at Göztepe Education and Research Hospital, Neurosurgery Clinic between September 2008 and December 2009 were prospectively reviewed. History and complaints were obtained from the case reports. Neuroradiological evaluation was performed with magnetic resonance imaging. Surgical findings of cases were reported in the operation notes. Microscopic posterior hemipartial laminectomy and discectomy were performed in all cases. Degenerated herniated disc material of all cases extracted during surgery was evaluated with immunohistochemical staining in Marmara University, Institute of Neurological Sciences, Pathology Laboratory., Results: Comparing the immunohistochemical staining of cases who were 50 years or younger and cases who were over 50 years old, statistical significance was determined., Conclusion: Matrix metalloproteinase 11 has a role in degenerating intervertebral disc disease, but it is not the only factor. Matrix metalloproteinase 11 might be a genetic factor in young-middle aged patients.

Matrix metalloproteinases (MMPs) have been known to play a pivotal role in the age- and/or disease-related degradation of intervertebral discs. We aimed to explore as to whether the expression of these enzymes is correlated to disc degeneration caused by increasing age and severity of herniation in the East Asian population. Thus, we studied the expressions of MMP-1 (collagenase), MMP-2 (gelatinase) and MMP-14 (membrane-type protease) in 65 patients diagnosed with lumbar disc herniation. The patients were divided into 3 groups according to their age, and the severity of herniation was graded on the basis of magnetic resonance imaging (MRI). Immunohistochemistry analysis was conducted to determine the expression of different MMPs in the post-surgery disc specimens. The results showed that expressions of these three enzymes were directly and positively related to the degree of disc degradation. Whereas, the MMP-1 expression was found to be elevated with the increasing age, the MMP-2 and MMP-14 remained unchanged in groups of different ages. A direct correlation between the expressions of MMP-2 and MMP-14 suggested a role of MMP-14 in the modulation of MMP-2 expression.

The authors studied the nuclear magnetic resonance films and the expression of MMP-1 and TIMP-1 in disk specimens' of patients who had undergone operations for lumbar disk herniation. Forty-one lumbar disk patients were evaluated imaging for degenerative changes and their disk specimens immunohistochemical expression of MMP-1 and TIMP-1. The degree of degenerative changes was based on magnetic resonance imaging films. Sections of disk immunostained for MMP-1 and TIMP-1 were evaluated semiquantitatively. Patients were categorized in three age groups: <30 years, from 30 to 60 years, and >60 years of age. The expressions of MMP-1 and TIMP-1 were related to patients' age and degree of degenerative changes. There were statistical differences in the expression of MMP-1 and TIMP-1 between the age and degree of degenerative changes groups. With the degree of degenerative changes, the expression of MMP-1 and TIMP-1 increased obviously. But in old age group, the expression of MMP-1/TIMP-1 was higher than the young groups. The expressions of MMP-1 and TIMP-1 were strongly correlated to the age and the degree of the degenerative changes. An important finding in this study is the unbalance of the expression of MMP-1 and TIMP-1 along with the growth of the age.

Object: Disc-related disorders such as herniation and chronic degenerative disc disease (DDD) are often accompanied by acute or chronic pain. Different mediators have been identified in the development of radicular pain and DDD. Previous studies have not analyzed individual cytokine profiles discriminating between acute sciatic and chronic painful conditions, nor have they distinguished between different anatomical locations within the disc. The aim of this study was to elucidate the protein biochemical mechanisms in DDD., Methods: The authors determined expression levels of matrix metalloproteinase-3, transforming growth factor-β (TGF-β), tumor necrosis factor-α, interleukin-1α, and pro-substance P using enzyme-linked immunosorbent assay and Western blot analyses in patients suffering from DDD (n = 7), acute back pain due to herniated discs with radiculopathy (n = 7), and a control group (n = 7). Disc tissue samples from the anulus fibrosus (AF) and nucleus pulposus (NP) were analyzed. Statistical analysis was performed using nonparametric tests., Results: A distinct distribution of cytokines was found in different anatomical regions of intervertebral discs in patients with DDD and herniated NP. Increased TGF-β levels were predominantly found in DDD. Matrix metalloproteinase-3 was increased in acute herniated disc material. Increased levels of substance P were found in patients suffering from DDD but not in patients with disc herniation. The data showed significantly higher levels of proinflammatory cytokines in the AF and NP of patients with DDD, and the expression levels in the AF were even higher than in the NP, suggesting that the inflammatory response initiates from the AF., Conclusions: These results highlight the complex mechanisms involved during disc degeneration and the need to distinguish between acute and chronic processes as well as different anatomical regions, namely the AF and NP. They also highlight potential problems in disc nucleus replacement therapies because the results suggest a biochemical link between AF and NP cytokine expression.

One of the most treatable causes of lower back pain and associated sciatica is lumbar disc herniation (LDH), which is characterized by rupture of the hard outer wall (annulus fibrosis) in a lumbar intervertebral disc. In the current study, we aimed to: (1) develop and characterize a rat model of sciatica induced by LDH, while introducing a novel method of epidural catheterization; (2) use this model to evaluate the effect of osthole on pain due to LDH, and (3) gain insight into the mechanisms through which osthole affects sciatica induced by LDH. The results indicate that our newly developed rat model maintained mechanical allodynia for 28 days without reduction. Moreover, cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) were overexpressed in the associated inflammatory response, which is consistent with clinical manifestations of the disease. We then used this model to study the effect and mechanisms through which osthole affected pain due to LDH. Our study suggests that osthole is capable of reversing hyperalgesia due to LDH, potentially through modulation of activity of COX-2 and NOS, two important proteins for the exacerbation of pain due to LDH. Finally, a molecular modeling simulation showed that osthole has unique binding capabilities to both NOS and COX-2. As the model-induced mechanical hyperalgesia response was consistent, and the position of the catheter tip and the extension/spreading of the drug in the epidural space were reliable, this study developed an improved model to study remedies for sciatic pain. Moreover, our studies demonstrate that osthole may be a feasible treatment for the reduction of pain due to hyperalgesia., (Copyright © 2012 S. Karger AG, Basel.)

Intervertebral disc (IVD) degeneration suggests a complex process influenced by genetics, lifestyle and biomechanics, which accounts for the development of low back pain (LBP) and lumbar radiculopathy, a major cause of musculoskeletal disability in humans. The family of Akt/PKB kinases is a principal mediator in the signal transduction pathways, which contribute to transcriptional regulation, cell growth, proliferation, apoptosis, and survival ability. The purpose of this study was to evaluate the transcriptional profile of the AKT family genes in human herniated discs and the involvement of the PI3K-Akt signaling pathway in human IVD degeneration. Real-time PCR analysis was used to assess the mRNA expression pattern of the three Akt/PKB isoforms in 63 herniated and 10 control disc specimens. Our results showed a significant positive correlation between AKT1 and AKT3 mRNA in herniated discs suggesting a synergistic action between these isoforms in disc herniation. Interestingly, AKT2 mRNA was up-regulated in patients with acute pain during the first 12 months, indicating that AKT2 transcriptional activation may be associated with acute rather than chronic inflammation and phagocytosis. Finally, Akt1/PKB transcription presented a stepwise activation as disc herniation deteriorated. Our findings provide evidence on the transcriptional activation of the Akt/PKB pathway indicating that it is involved in lumbar disc degeneration. There is need for further studies to elucidate the exact role and down-stream signaling action of Akt/PKB isoforms in the pathogenesis of lumbar disc herniation.

Many studies have demonstrated that apoptosis is involved in the development of disc degeneration. The initiator caspase 9 is activated through the apoptosome-driven intrinsic apoptotic pathway. The present study aimed to assess the potential association between the caspase 9 gene polymorphism and lumbar disc herniation (LDH) susceptibility, as well as severe grades of disc degeneration in the Han population in northern China. Genotyping was performed using the polymerase chain reaction and polymorphism was analyzed by restriction endonuclease cleavage in 387 patients with LDH and 412 control subjects. The allelic frequencies of caspase 9 Ex5+32 A were 0.483 and 0.391 in case patients and control subjects, respectively. Compared to those with the AA genotype, subjects with the GA/GG genotype have a higher risk to develop LDH (odds ratio 1.91; 95% confidence interval 1.29-2.81). Moreover, the GA/GG genotype was found to contribute to the risk of more severe grades of disc degeneration, as observed in magnetic resonance imaging scan. In conclusion, this study suggests that the single nucleotide polymorphism in the caspase 9 Ex5 + 32 G/A may be associated with LDH and disc degeneration in the Han population of northern China.

The involvement of matrix metalloproteinases (MMPs) in both the pathogenesis of intervertebral disc (ID) herniation and the spontaneous regression of herniated ID fragments remains only partially elucidated. The purpose of the present study was to simultaneously examine the transcript levels of a large number of MMPs (-1, -3, -8, -9, -13 and -14) and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs) and to investigate their correlation with the clinicopathologic profile of patients suffering from symptomatic lumbar ID herniation. mRNA expression levels were determined by means of the real-time polymerase chain reaction in 63 herniated and 10 control ID specimens. Our results showed multiple positive correlations among all MMPs and ADAMTS-4 mRNA in herniated samples, indicating their possible synergistic effect in ID herniation. MMP-9 and -13 mRNA levels were significantly elevated in patients with chronic pain, presumably as a consequence of neovascularization and chronic inflammation. Smoking habits were found to have a negative dose-dependent effect on the transcript levels of MMP-3 and MMP-13 and a positive correlation with pain intensity, suggesting an unfavorable role for smoking in the regression process of herniated disc fragments. Our findings provide evidence of the molecular portrait of MMPs and ADAMTS-4 in lumbar ID herniation, as well as of its association with the clinicopathological profile of the patients included in this study, reinforcing the hypothesis of MMPs involvement in the natural history of ID herniation. However, further studies are necessary to elucidate the exact role of MMPs in the resorption process of herniated lumbar discs.

Objective: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs., Introduction: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs., Methods: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2) was evaluated using immunohistochemistry and real-time RT-PCR analysis., Results: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process., Conclusion: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1.

The disruption of the extracellular disc matrix is a major hallmark of disc degeneration. This has previously been shown to be associated with an up-regulation of major matrix metalloproteinase (MMP) expression and activity. However, until now hardly any data are available for MMP/TIMP regulation and thereby no concept exists as to which MMP/TIMP plays a major role in disc degeneration. The objective of this study was, therefore, to identify and quantify the putative up-regulation of MMPs/TIMPs on the mRNA and protein level and their activity in disc material in relation to clinical data and histological evidence for disc degeneration. A quantitative molecular analysis of the mRNA expression levels for the MMPs (MMPs-1, -2, -3, -7, -8, -9, -13) and the MMP inhibitors (TIMPs-1 and -2) was performed on 37 disc specimens obtained from symptomatic disc herniation or degeneration. In addition, disc specimens from patients without disc degeneration/herniation (=controls) were analyzed. Expression of MMPs-1, -2, -3, -7, -8, -9, -13 and TIMPs-1, -2 was analyzed using quantitative RT-PCR, normalized to the expression level of a house keeping gene (GAPDH). Gene expression patterns were correlated with MMP activity (in situ zymography), protein expression patterns (immunohistochemistry), degeneration score (routine histology) and clinical data. MMP-3 mRNA levels were consistently and substantially up-regulated in samples with histological evidence for disc degeneration. A similar but less pronounced up-regulation was observed for MMP-8. This up-regulation was paralleled by the expression of TIMP-1 and to a lesser extent TIMP-2. In general, these findings could be confirmed with regard to protein expression and enzyme activity. This study provides data on the gene and protein level, which highlights the key role of MMP-3 in the degenerative cascade leading to symptomatic disc degeneration and herniation. Control of the proteolytic activity of MMP-3 may, therefore, come into the focus when aiming to develop new treatment options for early disc degeneration.

Introduction: Spontaneous resorption of disc herniation (DH) after sciatica is well documented. The matrix metalloproteinases (MMP)-1 and MMP-3 are enzymes potentially involved in this process. Glucocorticoid injections are commonly used for treatment, and other anti-inflammatory molecules like tumor necrosis factor (TNF) inhibitors are under clinical investigation. However, little is known about the effect of these molecules on DH resorption., Methods: DH tissue was harvested from patients undergoing surgery for sciatica. Samples were thoroughly washed. Diced explants were cultured ex-vivo in 1) 0.5 ml Dulbecco's modified Eagle's medium (DMEM) 10% fetal calf serum (FCS), (controls), 2) recombinant interleukin 1 receptor antagonist (IL-1Ra), (100 ng/ml), 3) dexamethasone (10E-5 M), or 4) TNF inhibitor monoclonal antibody (10 microg/ml). Supernatants were harvested at 48 hours and frozen. Immunocapture activity assays determined total MMP activity, active MMP levels and pro-MMP levels., Results: Fourteen DH tissue samples were analysed. Levels of all forms of MMP-3 were higher than the respective levels of MMP-1(P < 0.01). In particular, the median (interquartile range [IQR]) total MMP-3 level was 0.97 (0.47 - 2.19) ng/mg of tissue compared to 0.024 (0.01 - 0.07) ng/mg of total MMP-1 level (P < 0.01). Incubation with IL-1Ra, dexamethasone, or TNF inhibitors significantly decreased levels of all forms of MMP-3 (P < 0.05). Dexamethasone significantly decreased the ratio of active MMP-3 to total MMP-3 activity. A significant inhibitory effect of dexamethasone was observed only on active MMP-1, while IL-1 and TNF inhibitor had no significant effect on any form., Conclusions: MMP-3 appears to play a greater role than MMP-1 in DH resorption. Dexamethasone, IL-1-Ra and TNF inhibitor decreased active MMP-3, indicating that the clinical use of these drugs may affect the resorption of DH under certain conditions.

Study Design: Proteinases were immunohistochemically stained to analyze degenerated discs and paradiscal tissues in comparison to contiguous control tissues in an experimental porcine model of intervertebral disc degeneration., Objective: The aim was to analyze plasmin and metalloproteinases known to participate in mutual activation cascades., Summary of Background Data: Comparison of the degenerated discs and paradiscal structures with control tissues disclosed accumulation of plasmin and induction of matrix metalloproteinases (MMP), MMP-1 and MMP-2 in the discs, but some other MMPs in reactive and remodeling tissues., Material and Methods: In 6 domestic pigs, the cranial L4 endplate was perforated to penetrate the nucleus pulposus. Three months later, the animals were killed and the experimental and the contiguous control vertebrae, complete with their intervertebral discs, were excised and subjected to histologic and immunohistochemical examinations., Results: Immunohistochemical analysis disclosed increased expression of MMP-1 and MMP-2 in the traumatized and degenerated intervertebral discs. Some MMPs were also induced in all paradiscal structures (bone marrow, vertebral bone, and spinal ligaments), or decreased in already scarred areas. The common denominator for all the anatomic sites studied was accumulation of plasmin., Conclusion: Fibroblast collagenase (MMP-1) and gelatinase A (MMP-2), capable of degrading native and denatured collagen, were induced in degenerating intervertebral discs. Use of an experimental model enabled demonstration that biomechanical destabilization and degeneration of the disc also affects all other paradiscal structures, which are subjected to proteolysis and/or reparative fibrosis apparently representing remodeling of the spine subjected to pathologic stress. Profiling of various MMPs and plasmin, known to participate in mutual activation cascades, suggests that plasmin could activate pro-MMP-1, pro-MMP-2, pro-MMP-3, pro-MMP-7, pro-MMP-9, and pro-MMP-13 and alone or/and in cooperation with MMP-3 initiate at least 2 mutual MMPs activation cascades driven by activated MMP-3 and MMP-7.

Study Design: Immunohistochemical and behavioral study using rat models of lumbar disc herniation and cauda equina syndrome., Objective: To investigate the expression of activated p38 mitogen-activated protein kinases (p38 MAP kinase; p38) in the spinal cord and to determine the effect of intrathecal administration of a specific p38 inhibitor on pain in a lumbar disc herniation model and on motor function and hypoalgesia in a spinal canal stenosis (SCS) model., Summary of Background Data: In pathologic lumbar disc herniation-induced neuropathic pain and compression of cauda equina-induced motor dysfunction and hypoalgesia caused by SCS, glia are activated and produce certain cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukins, which play a crucial role in the pathogenesis of nerve degeneration. p38 is phosphorylated by these cytokines, suggesting that it may play an important role in pain transmission and nerve degeneration. Here we have examined the role of p38 in rat models of lumbar disc herniation and SCS., Methods: Six-week-old male Sprague-Dawley rats were used. For the disc herniation model, autologous nucleus pulposus was applied to L5 nerve roots, which were then crushed. For the SCS model, a piece of silicon was placed under the lamina of the fourth lumbar vertebra. We assessed mechanical allodynia, hypoalgesia, and motor function using von Frey hairs, treadmill tests, and immunohistochemical localization of phosphorylated p38 (P-p38) in the cauda equina, dorsal root ganglion (DRG), and spinal cord, which were also double-stained with NeuN (neuronal marker), GFAP (astrocyte/Schwann cell marker), or isolectin B4 (IB4; microglia marker). We also examined the effects of intrathecal administration of a specific p38 inhibitor, FR167653, on nucleus pulposus-induced pain, hypoalgesia, and motor dysfunction following SCS., Results: We demonstrated that activated P-p38-immunoreactive cells in the spinal cord and cauda equina were not observed before nerve injury but appeared in the cauda equina, DRG, and spinal dorsal horn in the disc herniation and SCS models. Double-labeling revealed that most P-p38-immunoreactive cells were isolectin B4-labeled microglia and GFAP-immunoreactive Schwann cells. Intrathecal administration of the p38 inhibitor FR167653 decreased mechanical allodynia in the disc herniation model and improved hypoalgesia and intermittent motor dysfunction in the SCS model., Conclusions: Our findings suggest that activated p38 may play an important role in the involvement of microglia in the pathophysiology of pain following lumbar disc herniation and mechanical hypoalgesia, and motor nerve dysfunction of cauda equina following SCS.

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.

Study Design: Examination of ADAMTS-4 expression, and cellular lineages, distribution, and numbers of ADAMTS-4 (aggrecanase-1) expressing cells in herniated lumbar intervertebral discs., Objective: To determine the expression of ADAMTS-4, a metalloproteinase capable of digesting aggrecan, and its role in herniated lumbar intervertebral disc degradation., Summary of Background Data: Matrix metalloproteinases degrade extracellular matrix of herniated discs, but the mechanism of aggrecan degradation, the major component of intervertebral discs, is poorly understood., Methods: Surgically resected herniated lumbar intervertebral discs from 22 patients were subclassified into protrusion, subligamentous extrusion, transligamentous extrusion, and sequestration types. Reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemistry were used to evaluate ADAMTS-4 messenger ribonucleic acid and protein expression., Results: Expression of ADAMTS-4 messenger ribonucleic acid and protein was shown in the samples of herniated lumbar intervertebral discs. Immunohistochemical staining showed that ADAMTS-4 was mainly localized in CD68-positive mononuclear cells in granulation and adjacent disc tissues. ADAMTS-4 positive cell counts were significantly higher in transligamentous extrusion and sequestration than protrusion and subligamentous extrusion types. Alcian blue staining showed a decrease of proteoglycan in transligamentous extrusion and sequestration cases., Conclusions: Macrophages infiltrating granulation and adjacent disc tissues express ADAMTS-4, suggesting its involvement in herniated disc regression.

Objective: To detect matrix metalloproteinase (MMP)-9 in serum and CSF and determine relationships between MMP activity and severity of disease, duration of clinical signs, and duration of hospitalization in dogs with acute intervertebral disk disease (IVDD)., Animals: 35 dogs with acute IVDD and 8 clinically normal control dogs., Procedure: CSF and serum were collected from affected and control dogs. Zymography was used to detect MMP-9., Results: Activity of MMP-9 in CSF was detected in 6 of 35 dogs with IVDD; activity was significantly more common in dogs with duration of signs < 24 hours. Paraplegic dogs were more likely to have MMP-9 activity in the CSF than non-paraplegic dogs. No significant difference in hospitalization time was detected in dogs with IVDD between those with and without activity of MMP-9 in the CSF. Serum MMP-9 was detected more frequently in dogs with IVDD than in control dogs., Conclusions and Clinical Relevance: Data were consistent with results of experimental rodent spinal cord injury studies that indicate that MMP-9 is expressed early during secondary injury.

Degeneration of the intervertebral disc is an important clinical problem, which often contributes to low back pain. Since approximately 80-90% of the general population will be subject to back pain at some stage during their lifetime, this has major socioeconomic consequences. Matrix metalloproteinases (MMPs) have been implicated in the excessive breakdown of extracellular matrix components during disc degeneration. The aim of the present study was to evaluate the regulation of MMP-2 (gelatinase-A) and MMP-3 (stromelysin) produced by cultured ovine nucleus pulposus (NP) cells stimulated with interleukin-1beta (IL-1beta). NP cells were established in three-dimensional agarose culture and stimulated with IL-1beta under serum-free conditions. Conditioned media samples were evaluated by gelatin and casein zymography and by fluorimetry using an MMP-specific substrate. Time-course and dose dependencies were established for MMP-2, -3 production by the NP cells in response to the IL-1beta. Gelatin and casein zymography indicated that elevated levels of proMMP-2 and proMMP-3 were present in media samples in response to the IL-1beta treatment. After 24-96 h culture, levels of the active 43 and 45 kDa active MMP-3 were significantly elevated, whereas MMP-2 was present mainly as its 72 kDa pro-form. Additional 36, 28 and 21 kDa MMP species were also present after prolonged incubation with IL-1beta, probably representing MMP breakdown species. IL-1beta was a potent catabolic mediator for the NP cells, resulting in the production of elevated levels of MMP-2 and -3 in culture. However, approximately 70% of the MMP-2 was present as the 72 kDa pro-form, which suggests that some additional steps are involved in its activation in vivo.

Study Design: The expression of cyclooxygenase-2 was studied immunohistologically in specimens from lumbar disc herniation. The cultured disc cells also were examined to evaluate the significance of cyclooxygenase-2, which might be involved in the pathogenesis of lumbar disc herniation., Objective: To investigate whether cyclooxygenase-2 might be involved in the pathogenesis of lumbar disc herniation., Summary of Background Data: Prostaglandin E2 is one of the most important mediators contributing to pathogenetic components of lumbar disc herniation. Cyclooxygenase-2, the rate-limiting enzyme of prostaglandin E2 synthesis, has been identified and extensively investigated in other inflammatory diseases. However, the role of cyclooxygenase-2 in lumbar disc herniation has never been addressed., Methods: Fifteen specimens from patients with lumbar disc herniation and five control discs from traumatic burst fracture were harvested. The expression of cyclooxygenase-2 was evaluated immunohistologically. The ability of cultured disc cells to produce prostaglandin E2 with inflammatory stimulus in the presence or absence of a selective inhibitor of cyclooxygenase-2 was investigated. At the same time, the induction of cyclooxygenase-2 mRNA of these cells by reverse transcriptase-polymerase chain reaction was detected. The manner in which this prostaglandin E2 production could be suppressed by various doses of a cyclooxygenase-2 inhibitor also was investigated., Results: Immunohistologically, the expression of cyclooxygenase-2 was observed only in the lumbar disc herniation specimens. The cultured cells had a strong ability to produce prostaglandin E2 coinciding with cyclooxygenase-2 mRNA induction. A selective inhibitor of cyclooxygenase-2 inhibited this prostaglandin E2 production in a dose-dependent manner., Conclusion: Cyclooxygenase-2 might be involved in the pathogenesis of lumbar disc herniation through upregulation of prostaglandin E2 production.

This article reviews the role of metabolic factors, including metalloproteinases and cytokines, in the occurrence of degenerative disk disease and disk herniation. Given that mechanical factors alone cannot cause disk degeneration, studies must explore metabolic, genetic, nutritional, and age-related factors. Zinc metalloproteinases exert particularly important effects, not only directly, but also indirectly through promotion of neovascularization. The production of these enzymes is dependent on a number of cytokines and on the cell changes they induce. This complex effect acts both on disk matrix degeneration and on the pain generated by contact between the protruding disk and the nerve roots. However, it can have a favorable effect by promoting resorption of the herniated disk. Available data on the role for mechanical factors on the disk chondrocyte metabolism and on metalloproteinase production show that mechanical and metabolic factors interact closely to produce disk disorders.

Study Design: Herniated lumbar disc specimens were cocultured with peripheral blood mononuclear cells, and cells isolated from extruded disc were cultured to study the production of matrix metalloproteinases., Objective: To investigate the role of peripheral blood mononuclear cells infiltrating extruded discs and disc-derived cells in the production of matrix metalloproteinases., Summary of Background Data: Magnetic resonance imaging analysis of herniated disc patients revealed a progressive decrease in the size of herniated discs. Spontaneous regression of herniated disc is associated with infiltrating macrophages, and matrix metalloproteinases have been implicated in this phenomenon. However, the correlation between infiltrating macrophages and the production of matrix metalloproteinases has received little research attention., Methods: Each disc specimen was incubated with homologous peripheral blood mononuclear cells. The numbers of peripheral blood mononuclear cells attached to the surfaces of herniated discs were counted and the culture media was assayed for MMP-3. The cells isolated from herniated discs were incubated with cytokines and the production of matrix metalloproteinases was measured. Total RNA was extracted from herniated discs and RT-PCR was carried out., Results: Significantly larger numbers of peripheral blood mononuclear cells were attached to the surfaces of extruded discs, and higher amounts of MMP-3 were detected than those of control discs. The culture medium of extruded discs showed higher MMP-1 and MMP-3 production than those from controls. Significant enhancement of MMP-1 and MMP-3 mRNA expression was observed in the disc-derived cells stimulated with cytokines., Conclusion: These results suggest that peripheral blood mononuclear cells infiltrating extruded discs may secrete a variety of biologic materials capable of further recruiting monocytes into herniated discs in an autocrine fashion. Disc cells stimulated with cytokines showed enhanced production of matrix metalloproteinases, which might play an important role in spontaneous regression of disc materials.

Study Design: A comprehensive immunohistochemical study of matrix metalloproteinase activity in discs from patients with different disc diseases., Objectives: To identify individual matrix metalloproteinase enzymes that could contribute to the degeneration of the matrix of the intervertebral disc, to identify the cells that produce matrix metalloproteinases (for example, the endogenous disc cells or invading cells associated with vascularisation), and to determine if "aggrecanase" contributes to degradation of proteoglycans in disc disorders., Summary of Background Data: Matrix disorganization and loss of substance are the most common findings in degenerate discs, and proteinase enzyme activity is one means of causing these changes., Methods: Forty-nine discs from 46 patients with degenerative disc disease, posterior anular tears, spondylolisthesis, or disc herniation were studied immunohistochemically to determine the presence of matrix metalloproteinases 1, 2, 3, 7, 8, 9 and 13, tissue metalloproteinases 1 and 2, and proteoglycan degradation products generated by either matrix metalloproteinases or aggrecanase activity. In addition, in situ zymography was used to confirm matrix metalloproteinase activity., Results: The most extensive staining was seen for matrix metalloproteinases 1, 2, 3, and 9, with 91%, 71%, 65%, and 72% of samples having some immunopositivity for the respective antibodies. In contrast, staining for matrix metalloproteinases 7 and 8 was much less (38% for both). Tissue inhibitor of metalloproteinases 1 and 2 were expressed in 34% and 79% of specimens, respectively. Matrix metalloproteinases were found particularly in cell clusters and blood vessels of degenerate discs, with staining correlating positively with macroscopic degenerative grade. For all of the enzymes, there was most staining in the herniation specimens and least in the autopsy samples. The opposite was true of staining for the matrix metalloproteinases inhibitor, tissue inhibitor of metalloproteinases 2, with most found in the autopsy specimens. Enzyme activity was confirmed by in situ zymography and staining for matrix metalloproteinase degradation products of proteoglycans. In addition, there was staining with antibodies demonstrating aggrecanase degradation products., Conclusions: Matrix metalloproteinase activity is more prevalent in herniated discs than in other disc disorders studied, although matrix metalloproteinases may have been more common earlier in the disease progression. Matrix metalloproteinases can be produced by invading blood vessels and associated cells, as well as by indigenous disc cells. Aggrecanase activity, although present in some samples, was not as obvious as that of matrix metalloproteinases. In addition to altered matrix metalloproteinase production, there appears to be a change in the balance between enzymes and endogenous inhibitors, tissue inhibitors of metalloproteinases. This study highlights specific matrix metalloproteinases that might be most efficient to target in developing therapeutics for minimizing degradation of the extracellular matrix of the disc.

A study was conducted to analyze the distribution and diameter of muscle fiber types in samples of the medial paravertebral lumbar muscle, i.e., multifidus muscle, obtained from 76 patients who underwent surgery for disc herniation. The samples were compared with 41 control samples of corresponding muscle tissue taken from 41 young healthy subjects who had died a sudden death. Histochemical analysis of fibers associated with myofibrillar adenosine triphosphatase (ATPase) revealed the presence of Type I fibers (slow-twitch fibers) and of Type IIA and IIB fibers (fast-twitch fibers) in both the experimental and control samples. The respective percentage of muscle fibers was calculated and their diameters were measured. Type I fibers predominated in both groups and were significantly larger in diameter than Type IIA and IIB fibers. Both fast-twitch fiber types were distributed in almost equal proportions in the healthy women. In the healthy men, Type IIA fibers prevailed. In the healthy females, the percentage of Type I fibers was found to be slightly higher than in the males, but the diameter of all fiber types was respectively smaller. In the females who had undergone surgery, Type I fibers were significantly larger in diameter than those of the healthy subjects. On the other hand, the diameters of all muscle fiber types were significantly larger, and the percentage of both fast-twitch fibers were Idwer in the samples from men who underwent surgery, as compared to the healthy tissue samples. The morphometric changes in the multifidus muscle at the level of the protruded disc observed by the histochemical method for demonstration of myofibrillar ATPase could not be related to the compressed nerve root in the majority of cases in our study.

Herniated disc (HD) is a common health problem that is resolved by surgery unless spontaneous resorption occurs. HD tissue contains abundant macrophage infiltration and high levels of matrix metalloproteinases (MMPs) MMP-3 and MMP-7. We developed a model system in which disc tissue or isolated chondrocytes from wild-type or MMP-null mice were cocultured with peritoneal macrophages and used this system to investigate the role of MMPs and chondrocyte/macrophage interactions in disc resorption. We observed a marked enhancement of MMP-3 protein and mRNA in chondrocytes after exposure to macrophages. Chondrocytic MMP-3, but not MMP-7, was required for disc resorption, as determined by assaying for a reduction in wet weight and proteoglycan content after 3 days of coculture. Surprisingly, chondrocyte MMP-3 was required for the generation of a macrophage chemoattractant and the subsequent infiltration of the disc tissue by proteolytically active macrophages. We conclude that macrophage induction of chondrocyte MMP-3 plays a major role in disc resorption by mechanisms that include the generation of a bioactive macrophage chemoattractant.

Herniated disc (HD), one of the major causes of low back pain, is often resolved spontaneously without surgical intervention. Resorption is associated with a marked increase in infiltrating macrophages, and the matrix metalloproteinases (MMP) MMP-3 and MMP-7 have been implicated in this phenomenon. We developed a murine organ culture model in which intact intervertebral discs were cocultured with peritoneal macrophages to investigate the role of MMPs in HD resorption. Using macrophages isolated from MMP-null mice, we report that macrophage-produced MMP-7 was required for proteoglycan degradation, loss of wet weight, and macrophage infiltration of cocultured discs. The inability of MMP-7-deficient macrophages to infiltrate discs could not be attributed to a defect in macrophage migration. MMP-7 was required for the release of the cytokine TNF-alpha from peritoneal macrophages. The generation of soluble TNF-alpha was essential for the induction of MMP-3 in disc cocultures, which in turn is required for the generation of a macrophage chemoattractant and subsequent macrophage infiltration. TNF-alpha release from macrophages was necessary but insufficient for disc resorption, which required macrophage infiltration. We conclude that there is extensive communication between macrophages and chondrocytes in HD resorption and that an essential component of this communication is the requirement for MMPs to release soluble bioactive factors.

Chondrocytes may play an important role in the inflammatory reactions involving intervertebral discs: the role of metalloproteases (MMP) and the imbalance between proteases and their inhibitors seems to play a crucial role in the disc degenerative process. In the present study we have analysed the production of collagenase-1 and stromelysin in different parts of herniated intervertebral discs. Ten samples of herniated lumbar discs were obtained from adult patients operated on for primary microdiscectomy because of persistent radicular symptoms and subdivided into three samples: 1. The nearest to the nerve root in close contact with it, 2. the sample obtained from the intervertebral space, and 3. the sample obtained from the vertebral plates. The DNA content of each specimen was measured in order to obtain the best estimate of the cell content as well as to normalise the content of collagenase-1 and stromelysin found in the same tissues with respect to the number of cells. In 9 out of the 10 cases the DNA content was significantly higher in samples obtained from the intervertebral plates than in samples obtained near the nerve root or the intervertebral space. DNA content did not show any significant difference considering disc samples contiguous to the nerve root and samples obtained from the intervertebral space. The production of collagenase-1 did not show a specific pattern, being produced in similar quantities in the three disc compartments. The production of stromelysin was significantly lower in disc samples obtained from the intervertebral plates. In conclusion the results of the present study suggest firstly that in the herniated lumbar disc the reactivity and the production of pro-inflammatory agents is independent of DNA content, i.e. the cellularity of the sample, and secondly that activated cells in the intervertebral space and in the part of the disc in contact with the nerve root, even if scarce in number, are able to synthesise a proportionally higher quantity of MMP than the high-cellular disc fragments from the intervertebral plates, potentially playing a specific role in the inflammatory reactions.

Objectives: To assess the eventual presence, tissue localization, molecular forms, amount and activity of cathepsin G in the annulus fibrosus., Methods: Normal non-autolytic disc tissue was collected from cadavers within six hours after death. Degenerate disc samples were collected from low back pain patients undergoing anterior interbody fusion due to severe, discographically verified and painful disc degeneration, and from the posterior parts of intervertebral discs from 10 patients undergoing microscopic discoidectomy because of intervertebral herniation. Avidin-biotinperxidase complex staining of cathepsin G was quantitated by morphometry. Cellular localization was analyzed using double immunofluorescence staining of cathepsin G and CD68, proline 4-hydroxylase or von Willebrand factor. Neutral salt extracts were analyzed by using synthetic cathepsin G substrate in spectrophotometry, dot-immunoblotting and Western blotting., Results: Histological and morphometric image analysis showed increased cellularity, increased numbers of cathepsin G positive cells and neovascularization in degenerated discs compared to control discs. Neutral salt extract of disc tissue, degenerated or normal, in contrast to control material from synovial capsular tissue, did not contain measurable cathepsin G activity, although immunoreactive enzyme was detected in dot-immunoblotting. Western blotting demonstrated that the discal cathepsin G had an apparent molecular weight of 27 kDa., Conclusion: Due to its properties and localization in normal and pathologically altered tissue, cathepsin G probably plays both a direct and an indirect role in extracellular matrix degradation in the annulus fibrosus. Extracted cationic cathepsin G was immunoreactive, but was functionally inhibited by serpins or, more likely, by polyanionic proteoglycans and saccharins derived from the connective tissue matrix of the annulus fibrosus.

Study Design: A review of the current literature on the role of matrix metalloproteinases in intervertebral disc degeneration., Objective: To detail the characteristics of matrix metalloproteinases (classification, structure, substrate specificity and regulation) and to report previous studies of intervertebral discs., Summary of Background Data: Degeneration of the intervertebral disc, a probable prerequisite to disc herniation, is a complex phenomenon, and its physiopathologic course remains unclear. Matrix metalloproteinases probably play an important role but have received sparse attention in the literature., Methods: A systematic review of studies reporting a role of matrix metalloproteinases in intervertebral disc degeneration., Results: In several studies, investigators have reported the presence of proteolytic enzymes from disc culture systems and disc tissue extracts in degenerated human intervertebral discs, especially collagenase-1 (MMP-1) and stromelysin-1 (MMP-3). The matrix metalloproteinases are regulated by specific inhibitors (tissue inhibitors of metalloproteinases, or TIMPS), cytokines (interleukin-1), and growth factors., Conclusions: This field of application is of particular interest because conventional treatments are disappointing in chronic low back pain. Clinical trials with specific inhibitors of metalloproteinases are beginning in osteoarthritis.

The pathophysiologic mechanisms of painful radiculopathy caused by a herniated intervertebral disc remain unknown. This study sought to determine whether the autologous intervertebral disc produces pain related behavior and whether phospholipase A2 and nitric oxide are involved in the pathophysiologic mechanism producing the behavior. A rat model, in which autologous intervertebral discs were implanted on the nerve root in the lumbar spine, was used to measure hyperalgesia, which is a pain related behavior in the rat. In this experimental model, autologous nucleus pulposus and anulus fibrosus transplanted to lumbar nerve roots produced mechanical and thermal hyperalgesia, respectively. Epidural injection of a selective inhibitor for phospholipase A2 resulted in the disappearance of hypersensitivity to noxious mechanical stimuli. Thermal hyperalgesia produced by application of the anulus fibrosus was abated and abolished by epidural injections of saline and one of the inhibitors for nitric oxide synthase, respectively. The authors suggest that chemical mediators such as phospholipase A2 and nitric oxide, induced by extruded or sequestrated intervertebral discs, are involved in the pathophysiologic mechanisms of painful radiculopathy in lumbar disc herniations. This study may be useful in attempting to develop new medical approaches for treatments of lumbar disc herniation.

Study Design: Surgically obtained herniated lumbar disc specimens were stained with hematoxylin-eosin or toluidine blue (for detection of proteoglycans) or were immunostained with monoclonal antibodies (CD68), antihuman interstitial collagenase (matrix metalloproteinase [MMP]-1) and antihuman stromelysin (MMP-3)., Objective: To investigate the possible correlation of matrix metalloproteinase activity to granulation tissue formation and lumbar disc herniation, depending on the type of herniation., Summary of Background Data: Interstitial collagenase and stromelysin have been implicated in the degradation of the matrix of articular cartilage in rheumatoid arthritis, osteoarthritis and degenerated disc tissues. However, their role in the herniation of the intervertebral disc has received little study., Methods: Twenty-one specimens of lumbar disc herniation (classified as protrusions, subligamentous extrusions, transligamentous extrusions, and sequestrations) and four nonherniated discs were stained with hematoxylin-eosin or toluidine blue or were immunostained with monoclonal antibodies to CD20, CD45RO, and CD68, anti-MMP-1, and anti-MMP-3, using the avidin-biotin-peroxidase complex method. The amount of granulation tissue and results of staining were graded to examine differences in histology among the four herniation types., Results: In sequestration and transligamentous extrusion specimens, granulation tissue containing many CD68-positive macrophages was commonly observed. Most cells in granulation tissue, as well as chondrocytes, stained positively with anti-MMP-1 and anti-MMP-3 antibodies. Granulation tissue was less commonly observed in subligamentous extrusions and was absent from most protrusion specimens and all nonherniated specimens. B and T lymphocytes could not be demonstrated in granulation tissue., Conclusions: The increased staining of MMP-1 and MMP-3 associated with inflammatory cells of granulation tissue in herniated discs suggests a causal correlation of these proteinases to tissue degradation in herniation.

Phospholipase A2 (PLA2) has been suggested to be present in herniated disc tissue and it could possibly be involved in sciatica/ discogenic back pain mechanisms. In the present study the occurrence of two different phospholipase A2 enzymes, (1) low molecular weight (14 kDa) group II synovial-type (sPLA2) and (2) high molecular weight (85 kDa) group IV cytosolic (cPLA2), were compared. Fifty-three disc prolapses obtained at disc operations were analyzed by immunohistochemistry, using anti-human monoclonal antibodies to sPLA2 and cPLA2, respectively. Only cell-associated (disc cells, hyaline cartilage chondrocytes) sPLA2 and cPLA2 immunoreactivity could be observed. The results showed that sPLA2 was more common (25/53, 47%) than cPLA2 (13/53, 25%). sPLA2 and cPLA2 were simultaneously present in 13 of 53 samples (25%). However, both PLA2 enzymes were predominantly present in hyaline cartilage cells (sPLA2: 16/53, cPLA2: 5/53), being less commonly observed in disc cells (sPLA2: 6/53, cPLA2: 3/53). In addition, three samples for sPLA2 and two samples for cPLA2 exhibited immunoreactivity in cartilage and disc cells simultaneously. sPLA2 was observed in no other locations, but in 3 of 53 samples cPLA2 was observed more diffusely in areas of granulation tissue, possibly in macrophages. No gender- or age-related dependence for either type of PLA2 enzyme immunoreactivity could be observed. Neither did their occurrence relate to clinical data such as straight leg raising or neurological deficit. The results do not support a major role for either of the two disc-cell-associated PLA2s in disc pathophysiology. For both enzymes, the major pool appears to reside in cartilage tissue cells, presumably in dislodged end-plate fragments. Disc cells are apparently unlikely candidates for major PLA2 storage.

To assess its possible role in the pathophysiology of intervertebral disc degeneration, we investigated the production of matrix metalloproteinase-3 (MMP-3) using human intervertebral disc explant culture. Five normal and 10 degenerated disc specimens were used. The levels of MMP-3 released in the medium were measured with use of an enzyme immunoassay. The results showed that the level of MMP-3 in the degenerated group (0.57 microg/ml/mg wet weight; n = 10) was significantly higher than that of the control group (0.29 microg/ml/mg wet weight; n = 5) (p < 0.05). Immunostaining of MMP-3 revealed that the ratio of positive staining cells in the degenerated group was greater than that of the control group. These observations suggest that MMP-3 produced by human intervertebral disc may be involved in the intervertebral disc degeneration, particularly in the initiation of matrix degradation of intervertebral disc.

Changes in the macromolecular matrix of the intervertebral disc may predispose to biomechanical failure of the disc. Such changes would involve extracellular enzymes capable of altering the collagen and proteoglycan of the disc matrix. In this study, tritium-labeled type I collagen was used as a substrate to estimate the activity of collagenase in the discs of 41 cases of lumbar disc protrusion (LDP) patients by surgical intervention. The annulus fibrous (AF) and nucleus pulposus (NP) were measured separately. 34 normal discs harvested by autopsy acted as controls. For estimation of relative neutral proteinase content of 6 normal and 16 degenerated lumbar discs, polyacrylamide gelelectrophoresis (PAGE), heat-denatured collagen as a substrate, and photo-density scanning with peak area autocalculating system were adopted. The results presented that both AF and NP of the normal discs had a similar lower collagenolytic activity and a very limited activity of neutral proteinase, while the degenerated discs showed a higher activity, especially in the degenerated NP. The extruded type of LDP got a higher collagenolytic activity in NP than that of the prolapsed LDP. The fact showed that the matrix degrading enzymes play a very important role in the process of lumbar disc degeneration. The difference of disc degeneration is the biochemical basis of different clinical types of LDP. Matrix degrading enzyme system is a very complexed multienzymatic system. Other neutral proteinases may join this system besides the collagenase.

Study Design: Prospective study of phospholipase A2 activity in the serum and intervertebral discs of patients undergoing surgery for sciatica due to disc herniation., Objectives: To determine correlations between herniated disc phospholipase A2 and clinical, radiographic, and anatomic signs of common sciatica; to evaluate serum phospholipase A2 activity as a marker of disc phospholipase A2; and to investigate the in vivo effect of piroxicam on disc phospholipase A2., Summary of Background Data: Several studies suggest disc inflammation as a mechanism of sciatica due to disc herniation, and phospholipase A2 emerges as a key enzyme of cartilage and disc tissues., Methods: Phospholipase A2 activity was determined, using the degradation of a specific substrate, in the serum and discs of 31 patients (14 treated with acetaminophen and 17 treated with piroxicam) undergoing surgery for sciatica due to lumbar disc herniation. Visual analog scale for pain, Dallas Pain Questionnaire, Lasègue's sign, radiographic stage of degeneration of the herniated disc, volume of disc herniation shown by computed tomography, and surgical findings were recorded., Results: Disc phospholipase A2 activity was independent of the patient's age or sex, the radiologic stage of disc degeneration, and the volume of the herniation, and showed no significant correlation with Lasègue's sign or pain measured on a visual analog scale. The correlation between disc phospholipase A2 and the Dallas category of items measuring the impact of pain on daily activities approached the level of significance (P = 0.07). Disc phospholipase A2 activity was significantly higher in cases of sequestrated discs than in other herniations. Disc phospholipase A2 was significantly correlated with serum phospholipase A2, and was significantly lower in patients treated with piroxicam than in those treated with acetaminophen., Conclusions: Disc phospholipase A2 is thought to participate in the physiopathology of sciatica and to bemodulated by nonsteroidal anti-inflammatory drug therapy. Serum phospholipase A2 is suggested as a biologic marker of disc inflammation in patients with sciatica.

Previously we reported that PGBx, a prostaglandin oligomer with anti-inflammatory activity, inhibited 14 kDa phospholipase A2 (PLA2) activity and blocked arachidonic acid mobilization in prelabeled human neutrophils (Biochim. Biophys. Acta 1006:272-277, 278-286, 1989) This study describes a new inhibitor of phospholipase A2, PX-52, that also blocks agonist induced arachidonic acid mobilization in prelabeled cells. PX-52, a fatty acid polymer, inhibited hydrolysis of 14C-oleate labeled E.coli by a variety of 14 kDa PLA2s including human PMN, sperm, synovial fluid and disc, as well as porcine pancreas, N. naja, and bee venom in a dose-dependent manner with IC50s ranging from 1.0-3.7 uM. Inhibition of activity was comparable at different Ca2+ concentrations, but was relieved by increasing substrate concentration or by methylation of PX-52. Hydrolysis of [14C]-arachidonyl phosphatidylcholine by 85 kDa, cytosolic PLA2 from U937 cells was similarly inhibited by PX-52, the IC50 = 5 uM. Arachidonic acid mobilization induced by A23187 in prelabeled human PMNs was blocked by PX-52; IC50 = 10-15 uM while concentrations of up to 80 uM oleate had no effect. These results demonstrate that PX-52 inhibits the in vitro activity of secretory and cytosolic PLA2s and agonist-induced arachidonic acid release from human cells. Given its ability to block the arachidonic acid cascade, PX-52 may be useful in the control of inflammation.

Study Design: Group II phospholipase A2 enzyme activity was studied biochemically and immunohistochemically in tissue samples from disc prolapses, degenerated discs, and macroscopically normal discs. In parallel, phospholipase A2 and inflammatory cells were studied by indirect immunocytochemistry., Objectives: To compare phospholipase A2 activity in normal discs and abnormal discs by an identical assay for phospholipases A2, and to compare the occurrence of inflammatory cells with phospholipase A2 activity and immunoreactivity., Summary of Background Data: It has been suggested that a high phospholipase A2 enzyme activity in herniated disc tissue could be significant in abnormal states such as sciatica and discogenic pain. No comparison between healthy disc tissue and samples of abnormal discs (degenerated or herniated) has been carried out. In particular, an identical assay for phospholipase A2 for such tissue samples, supported by immunohistochemical staining data, has never been applied in parallel to normal and abnormal disc tissue, and neither have such results been compared with the demonstration of inflammatory cells., Methods: Group II phospholipase A2 enzyme activity was determined, in parallel, using an identical assay for tissue samples from 11 macroscopically normal discs, 33 disc herniations, and six discs showing degeneration by discography. For determination of phospholipase A2 enzyme activity, a radioassay using 1-palmitoyl-2-(1-14C)linoleoyl-L-3-phosphatidylethanolamine as the phospholipid substrate was used. Total tissue DNA as an estimate of total tissue cell number was measured in parallel with phospholipase A2 activity. All tissue samples also were studied by indirect immunocytochemistry, locating phospholipase A2 and T and B lymphocytes., Results: Neither degenerated nor herniated disc tissue samples demonstrated a higher phospholipase A2 activity than control disc tissue samples. Average phospholipase A2 activity was actually higher in the control samples than in herniated disc samples (Mann-Whitney test, P < 0.001), possibly a result of a higher total DNA (P < 0.005). The observed level of phospholipase A2 activity was lower than that of inflammatory human synovial fluid. Neither was there marked immunoreactivity for phospholipase A2, which was observed in chondrocytes in areas of cartilage and occasional disc cells, supporting the biochemical results. Lymphocytes were more numerous only in herniated disc samples (15%), and their presence showed little overlap with phospholipase A2 immunoreactivity., Conclusions: Synovial-type (Group II) phospholipase A2 enzyme activity is not particularly high in disc tissue and does not appear to be higher in herniated or degenerated discs than control disc tissue. Immunoreactivity to phospholipase A2 is seen only occasionally and is strong only when cartilage tissue is present. Neither are inflammatory lymphocytes commonly observed.

We measured activities of dynorphin-converting enzyme (DCE), substance P endopeptidase (SPE) and angiotensin-converting enzyme (ACE) in cerebrospinal fluid (CSF) in 13 patients with rhizopathic pain from an herniated lumbar disc, in 9 patients with pain from coxarthrosis and in 11 control patients without pain. In the patients with disc hernia and coxarthrosis, another sample of CSF was analyzed 3-12 months after treatment, when pain had subsided. The DCE activity in the patients was higher than that in both the control patients and the patients with pain from coxarthrosis (nociceptive pain). Similarly, the activity of SPE was lower in the patients with herniated lumbar disc than in controls and in the patients with coxarthrosis. After treatment, the difference in activity compared to controls was lower, but still significant in patients with herniated discs. The ACE activity did not differ from controls in patients with ischialgia, while it was increased in patients with coxarthrosis. This increase also remained after arthroplasty with pain relief. In conclusion, measurements of neuropeptides may be useful for evaluating neuropathic pain.

Study Design: Herniated cervical disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced., Objectives: Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in cervical intervertebral disc degeneration and in the pathophysiology of cervical radiculopathy., Summary of Background Data: Neck pain with or without radiculopathy is a common clinical problem, but the etiology of neck pain and the exact pathophysiology of radiculopathy remain uncertain. We have previously reported the production of various biochemical agents by herniated lumbar disc specimens in vitro. Because of a lack of such studies in the literature with respect to the cervical spine, the purpose of this study was to determine whether similar biochemical agents of inflammation and tissue degradation were being produced by herniated cervical disc specimens., Methods: Eighteen herniated cervical discs were obtained from 15 patients undergoing anterior disc surgery. The specimens were cultured and incubated for 72 hours, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, six cervical discs specimens were obtained from three patients undergoing anterior surgery for traumatic burst fractures, and similar biochemical analyses were performed., Results: The culture media from the herniated cervical disc specimens showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated disc specimens compared with the control discs. Interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of the herniated or control discs., Conclusions: Herniated cervical disc specimens were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These results were similar to those obtained in herniated lumbar disc specimens that we have previously reported. These products may be intimately involved in the biochemistry of disc degeneration and the pathophysiology of radiculopathy.

Neutral proteinases were partially purified from human intervertebral disc by extraction with guanidine hydrochloride followed by Sephadex G-75 column chromatography. They showed gelatinolytic and elastolytic activities at neutral pH. The apparent molecular weights of these enzymes were 70 KD and 25 KD, the former being the complexed form of the latter. The sensitivity to various synthetic inhibitors indicated these enzymes to be serine elastases. Furthermore, they were considered to be leukocyte elastases because anti-leukocyte elastase antibody inhibited the proteinase activity of these enzymes. Proteinase activity was not detected in normal discs, except for some slight activity in the end-plate. In contrast, the degenerated discs showed high activity, especially in the nucleus pulposus and end-plate. Similar high levels of proteinase activity were found in the vertebral body adjacent to normal as well as degenerated discs. These findings suggest that leukocyte elastase, which is normally present in the vertebral body, flows into and degrades the matrix of the intervertebral disc under pathologic conditions.

The effect on human herniated intervertebral disc phospholipase A2 (HD-PLA2) of a number of retinoids, antirheumatic drugs and reported PLA2 inhibitors was evaluated using autoclaved [1-14C]-oleate-labeled Escherichia coli membranes as the substrate. Dexamethasone, non-steroidal antiinflammatory drugs, aristolochic acid and retinol were inactive, whereas a marked inhibition was found for manoalide, retinal, nordihydroguaiaretic acid and p-bromophenacyl bromide after preincubation with the enzyme (IC50 values 0.25, 4, 5 and 5 microM, respectively). The results are parallel to those obtained with the PLA2 purified from human synovial fluid.

In a prospective study of 251 patients operated upon for lumbar disc herniation it has been investigated whether the preoperative and early postoperative values of Elastase-alpha-1 Proteinase Inhibitor (EPI)-an indicator of inflammatory processes-and C-reactive Protein (CRP)-a well known predictor of some postoperative complications-were correlated to the later development of discitis. Postoperatively discitis developed in 14 patients. A randomly chosen group of 15 complication-free patients out of the total of 251 cases was used as control group. Elevated EPI plasma values, especially in the pre-operative and first postoperative days, turned out to be significantly related to the likelihood of later discitis development, but no such relation for the CRP plasma values could be established. Thus and early prediction of patients at risk for this complication seems to be possible by pre- and postoperative measurement of EPI. It could be justified-but its usefulness has yet to be proven-to give antibiotics prophylactically and other anti-inflammatory medication in patients with elevated pre- and postoperative EPI values.