Lumbar Disc Tissue Research Articles

Lipid Peroxidation, Reduced Glutathione, and Glutathione Peroxidase Levels in Intervertebral Discs of Patients with Lumbar Degenerative Disc Disease.

BACKGROUND Either a reduction in antioxidant levels or an accumulation of reactive oxygen species can heighten susceptibility to oxidative damage in disc cells. To date, no research has investigated the levels of lipid peroxidation products (thiobarbituric acid reactive substances [TBARs]), reduced glutathione (GSH), and glutathione peroxidase (GPx) in excised human lumbar disc tissues affected by degenerative disease. Therefore, this study aimed to evaluate lipid peroxidation products in excised disc tissues from patients with degenerative disc disease. MATERIAL AND METHODS Forty-two patients were enrolled. Patients were divided into lumbar disc degeneration (LDD) and nonlumbar disc degeneration (nonLDD) groups according to Pfirrmann classification. Intervertebral discs were obtained from all patients during the operation and were homogenized for analysis. TBARs levels were measured using fluorometry. GSH levels and GPx activity were quantified spectrophotometrically using a kinetic method. RESULTS TBARs levels in excised discs from LDD patients (5.18±4.14) were significantly higher than those from nonLDD patients (2.56±1.23, P=0.008). The levels of TBARs tended to increase with the severity of degeneration according to the Pfirrmann classification. However, these 2 groups showed no significant differences in reduced glutathione levels or glutathione peroxidase activity (P>0.05). Patients with LDD exhibited a worse health-related quality of life, reflected in lower utility and EQ-VAS scores and higher Oswestry disability index scores. CONCLUSIONS There was a notable increase in lipid peroxidation products in the excised intervertebral discs of patients with LDD. This finding suggests that oxidative stress may contribute to the development of disc degeneration.

Histological analysis of nucleus pulposus tissue from patients with lumbar disc herniation after condoliase administration.

Condoliase is an enzyme used as a treatment for lumbar disc herniation (LDH). This enzyme degrades chondroitin sulfate (CS) in the nucleus pulposus of the intervertebral disc (IVD). However, there are cases in which symptoms do not improve, despite condoliase administration. This study reports histological analysis of lumbar disc tissue of LDH patients who underwent surgery because condoliase had no therapeutic effect. Between March 2019 and August 2019, 12 LDH patients who underwent full endoscopic spine surgery (FESS) discectomy at the Dezawa Akira PED Clinic were the subjects of the study. There are two study groups: six cases underwent FESS after condoliase administration, while six underwent FESS without condoliase administration. The average duration from drug administration to surgery was 152 days. Herniated disc removed at surgery was evaluated by histological staining including immunohistochemistry by anti-CS antibodies. Multiple large clusters (40-120 μm in diameter) were observed in the nucleus pulposus of those who received condoliase, but no clusters were observed in those who did not. The lumbar disc tissues, including the nucleus pulposus of recipients, were stained with anti-CS antibodies that recognize the CS unsaturated disaccharide, but non-administration tissue was not stained. These findings suggest that the enzyme acted on the nucleus pulposus, even in cases where symptoms were not improved by condoliase administration. Furthermore, there was no histological difference between stained images of the extracellular matrix in those who did or did not receive condoliase, suggesting that condoliase acted specifically on CS in the nucleus pulposus. We demonstrated that CS in the nucleus pulposus was degraded in patients in whom condoliase did not have a therapeutic effect. Moreover, condoliase acts in human IVD without causing necrosis of chondrocytes and surrounding tissues.

The clinical significance and function of miR-146 in the promotion of epidural fibrosis.

Epidural fibrosis is the main cause of failed back surgery syndrome. To investigate the role of miR-146 in the diagnosis and development of epidural fibrosis. Lumbar disc tissues were collected from 72 lumbar disc herniation patients (45 developed epidural fibrosis and 27 did not). The expression of miR-146 in collected tissues and isolated epidural fibroblasts was detected by RT-qPCR. The relative levels of pro-inflammatory cytokines were analyzed by ELISA. The effect of miR-146 on the proliferation of fibroblasts was evaluated by MTT assay. miR-146 was significantly upregulated in epidural fibrosis patients compared with control patients. The expression of miR-146 was closely associated with the location, lower limb symptom and duration of disease of epidural fibrosis patients, and was positively correlated with the relative levels of pro-inflammatory cytokines. Moreover, miR-146 could discriminate epidural fibrosis patients from control patients. In isolated epidural fibroblasts, the overexpression of miR-146 dramatically enhanced its proliferation and the inflammatory response. miR-146 serves as a diagnostic biomarker for the early detection of epidural fibrosis. The upregulation of miR-146 enhanced the fibroblasts proliferation and inflammatory response in epidural fibrosis. This study provides a novel potential therapeutic target for epidural fibrosis.

88. Cell stiffness decreases with severity of disc degeneration and inflammatory stimulation

88. Cell stiffness decreases with severity of disc degeneration and inflammatory stimulation

The correlation between microvessel pathological changes of the endplate and degeneration of the intervertebral disc in diabetic rats.

[This retracts the article DOI: 10.3892/etm.2012.868.].

Long non-coding RNA zinc finger antisense 1 expression associates with increased disease risk, elevated disease severity and higher inflammatory cytokines levels in patients with lumbar disc degeneration.

This study aimed to investigate the correlation of long noncoding RNA zinc finger antisense 1 (lncRNA ZFAS1) expression with disease risk, disease severity and inflammatory cytokines levels in lumbar disc degeneration (LDD) patients.83 LDD patients underwent surgery and 28 traumatized, non-LDD patients underwent lumbar disc surgery (controls) were consecutively enrolled in this case-control study. Lumbar disc tissue was obtained during surgery and herniated nucleus pulposus (HNP) was isolated to detect lncRNA ZFAS1 expression and inflammatory cytokines mRNA levels by RT-qPCR, and determine protein levels of inflammatory cytokines by western blot.HNP lncRNA ZFAS1 expression in LDD patients was up-regulated compared with controls (P < .001), and receiver operating characteristic (ROC) curve showed lncRNA ZFAS1 expression disclosed a good predictive value for LDD risk with area under curve (AUC) 0.753 (95% CI 0.646–0.859). And after adjustment by age, gender and body mass index (BMI), lncRNA ZFAS1 (P = .017) remained to be an independent predictive factor for higher LDD risk. In addition, lncRNA ZFAS1 expression was positively associated with Modified Pfirrmann Grade (P = .015). As to inflammatory cytokines, lncRNA ZFAS1 expression was observed to be positively correlated with TNF-α (P = .002), IL-1β (P = .007) and IL-6 (P = .015) mRNAs expressions while reversely associated with IL-10 mRNA level (P = .014); and lncRNA ZFAS1 expression was also positively correlated with protein levels of TNF-α (P = .038) and IL-6 (P = .027) while reversely associated with IL-10 protein expression (P = .039).lncRNA ZFAS1 expression associates with increased risk, elevated disease severity and higher inflammatory cytokines levels in LDD patients.

Physical disruption of intervertebral disc promotes cell clustering and a degenerative phenotype

To test the hypothesis that physical disruption of an intervertebral disc disturbs cell-matrix binding, leading to cell clustering and increased expression of matrix degrading enzymes that contribute towards degenerative disc cell phenotype. Lumbar disc tissue was removed at surgery from 21 patients with disc herniation, 11 with disc degeneration, and 8 with adolescent scoliosis. 5 μm sections were examined with histology, and 30-µm sections by confocal microscopy. Antibodies were used against integrin α5beta1, matrix metalloproteinases (MMP) 1, MMP-3, caspase 3, and denatured collagen types I and II. Spatial associations were sought between cell clustering and various degenerative features. An additional, 11 non-herniated human discs were used to examine causality: half of each specimen was cultured in a manner that allowed free ‘unconstrained’ swelling (similar to a herniated disc in vivo), while the other half was cultured within a perspex ring that allowed ‘constrained’ swelling. Changes were monitored over 36 h using live-cell imaging. 1,9-Di-methyl methylene blue (DMMB) assay for glycosaminoglycan loss was carried out from tissue medium. Partially constrained specimens showed little swelling or cell movement in vitro. In contrast, unconstrained swelling significantly increased matrix distortion, glycosaminoglycan loss, exposure of integrin binding sites, expression of MMPs 1 and 3, and collagen denaturation. In the association studies, herniated disc specimens showed changes that resembled unconstrained swelling in vitro. In addition, they exhibited increased cell clustering, apoptosis, MMP expression, and collagen denaturation compared to ‘control’ discs. Results support our hypothesis. Further confirmation will require longitudinal animal experiments.

Are radio-contrast agents commonly used in discography toxic to the intact intervertebral disc tissue cells?

In the literature, there have been no studies showing clear results on how radio-contrast pharmaceuticals would affect intact disc tissue cells. In this context, it was aimed to evaluate the effects of iopromide and gadoxetic acid, frequently used in the discography, on intact lumbar disc tissue in pharmaco-molecular and histopathological level. Primary cell cultures were prepared from the healthy disc tissue of the patients operated in the neurosurgery clinic. Except for the control group, the cultures were incubated with the indicated radio-contrast agents. Cell viability, toxicity and proliferation indices were tested at specific time intervals. The cell viability was quantitatively analysed. It was also visually rechecked under a fluorescence microscope with acridine orange/propidium iodide staining. Simultaneously, cell surface morphology was analysed with an inverted light microscope, while haematoxylin and eosin (H&E) staining methodology was used in the histopathological evaluations. The obtained data were evaluated statistically. Unlike the literature, iopromide or gadoxetic acid did not have any adverse effects on the cell viability, proliferation and toxicity (P<0.05). Although this study reveals that radio-contrast pharmaceuticals used in the discography, often used in neurosurgical practice, can be safely used, it should be remembered that this study was performed in an invitro environment.

The comparison of the mrna expressions of tgfbeta1, bfgf, igf-1, ngf and matrix metalloprotease iii genes in cervical and lumbar disc tissues

Aim: To evaluate and compare the mRNA expression of antioxidant genes between the cervical and lumbar degenerated disc tissues. Material and Methods: Obtained degenerated Nucleus Pulposus (DNP) materials were divided into two groups, which were cervical DNP (Group I) (n=20) and lumbar DNP (Group II) (n=20). There are 6 men and 14 women in group I with a mean age of 43.8 years and 13 men and 7 women in group II with the mean age of 40.3 years (28 to 54). All cervical DNP materials were obtained between C5 and C6 and all lumbar DNP materials were obtained between L4 and L5 levels. The TGFβ1, bFGF, IGF-1, NGF and Matrix metalloprotease III (MMP III) gene expressions of DNP materials were determined by polymerase chain reaction, Real-time polymerase chain reaction and western blotting techniques.Results: When the results of the two groups were compared by polymerase chain reaction, the expressions of TGFβ1, bFGF, IGF-1, NGF and MMP III was found lower in lumbar DNP group. Also the TGFβ1, bFGF, IGF-1, NGF, and Matrix metalloprotease III genes showed a decrease in gene expression levels when they were analyzed by Real-time polymerase chain reaction. Conclusion: These data showed that; decrease of the TGFβ1, bFGF, IGF-1, NGF and MMP III genes in the degenerated lumbar disc tissues, may related with the possibility of molecular background of the disease pathogenesis.

Transfection of lentivirus-bone morphogenetic protein 2 and lentivirus-inhibitor of differentiation 1 into nucleus pulposus for delaying intervertebral disc degeneration in an in vivo rabbit model

To investigate if the course of intervertebral disc degeneration (IDD) is delayed by injecting lentivirus (Lv) vector carrying bone morphogenetic protein 2 (BMP-2) and inhibitor of differentiation 1 (Id1) genes directly into the nucleus pulposus. Thirty-two New Zealand white rabbits, 2.0-2.5 kg in weight and 4 months in age, were used to establish the IDD models at L 3, 4, L 4, 5, and L 5, 6 discs with annular puncture via transabdominal approach. Thirty rabbits with successful modeling were randomly divided into 5 groups, 6 rabbits every group. At 4 weeks after modeling, rabbits were injected with Lv-BMP-2 (group A), with Lv-BMP-2 and Lv-Id1 (group B), with Lv-Id1 (group C), with Lv-green fluorescent protein (group D), and with PBS (group E). At 2, 4, and 8 weeks after injection, T2-mapping MRI was performed on 2 rabbits each group to obtain the T2 values, and then subsequently the lumbar disc tissues were harvested to test the mRNA expressions and contents of collagen type II and proteoglycan by real-time fluorescent quantitative PCR and ELISA methods. T2-mapping MRI demonstrated that there was no significant difference in the T2 value between different groups at immediate and 2 weeks after injection ( P>0.05). The T2 value of groups A and B was significantly higher than that of groups C, D, and E at 4 weeks after injection ( P<0.05), but no significant difference was observed between group A and group B ( P>0.05). The T2 value of group B was significantly higher than that of the other groups at 8 weeks after injection ( P<0.05). The real-time fluorescent quantitative PCR and ELISA showed that the expressions and contents of collagen type II and proteoglycan in group B were significantly higher than those in the other groups at 2, 4, and 8 weeks after injection ( P<0.05). Combined application of Lv-BMP-2 and Lv-Id1 can delay IDD changes in rabbit IDD models.

P120-Catenin Protects Endplate Chondrocytes From Intermittent Cyclic Mechanical Tension Induced Degeneration by Inhibiting the Expression of RhoA/ROCK-1 Signaling Pathway.

The changes of endplate chondrocytes induced by intermittent cyclic mechanical tension (ICMT) were observed by realtime reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis. To investigate the role of RhoA/ROCK-1 signaling pathway and E-cadherin/P120-catenin complex in endplate chondrocytes degeneration induced by ICMT. ICMT can induce the endplate chondrocyte degeneration. However, the relationship between P120-catenin or RhoA/ROCK-1 signaling pathway and endplate chondrocytes degeneration induced by ICMT is not clear. ICMT (strain at 0.5 Hz sinusoidal curve at 8% elongation) was applied to rat endplate chondrocytes for 6 days, 16 hours a day. The cell viability and apoptosis were examined by the LIVE/DEAD assay and flow cytometry. Histological staining was used to examine the lumbar disc tissue morphology and extracellular matrix. To regulate RhoA/ROCK-1 signaling pathway and the expression of E-cadherin and P120-catenin, RhoA/ROCK-1 pathway-specific inhibitors, E-cadherin, and p120-catenin plasmid were applied. Coimmunoprecipitation was employed to examine the interaction between E-cadherin and P120-catenin, P120-catenin, and RhoA. The related gene expression and protein location was examined by realtime reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. There was no change of viability verified by LIVE/DEAD assay and flow cytometry after ICMT loading. ICMT loading led to RhoA/ROCK-1 signaling activation and the loss of the chondrogenic phenotype of endplate chondrocytes. Inhibition of RhoA/ROCK-1 signaling pathway significantly ameliorated the degeneration induced by ICMT. The expression of P120-catenin and E-cadherin were inhibited by ICMT. ICMT reduced the interaction between P120-catenin and E-cadherin. Furthermore, over-expression of P120-catenin and E-cadherin can suppress the expression of chondrogenic gene, over-expression of P120-catenin can suppress the RhoA/ROCK-1 signaling pathway, but over-expression of E-cadherin cannot do it. P120-catenin protects endplate chondrocytes from ICMT Induced degeneration by inhibiting the expression of RhoA/ROCK-1 signaling pathway. N/A.

Increased Apoptosis, Expression of Matrix Degrading Enzymes and Inflammatory Cytokines of Annulus Fibrosus Cells in Genetically Engineered Diabetic Rats: Implication for Intervertebral Disc Degeneration

Introduction Diabetes mellitus is thought to be an important etiologic factor in intervertebral disc degeneration. However, what is little known is whether diabetes has an effect on annulus fibrosus (AF) cells. The purpose of our study was to investigate the effect of diabetes mellitus on apoptosis, expression of matrix degrading enzymes and inflammatory cytokines of AF cells in age-matched genetically engineered OLETF (diabetic) and LETO (control) rats. Material and Methods Lumbar disc tissues (L1–2 through L5–6) were obtained from 6-month old OLETF and LETO rats (10 each). We examined the AF cells and tissues using Masson trichrome stain, TUNEL, Western blotting, and reverse transcription polymerase chain reaction. The apoptosis index and the degree of expression of matrix degrading enzymes and inflammatory cytokines of AF cells were evaluated by semiquantitative method. Results OLETF rats showed increased body weight and abnormal 2-hour glucose tolerance tests compared with LETO rats. The apoptosis index and the degree of expression of Fas of AF cells were statistically higher in the OLETF rats. The degree of expression of matrix metalloproteinase-1, -2, -3 and -13 and tissue inhibitor of metalloproteinase-1 and -2 was statistically higher in the OLETF rats. The expression of interleukin-1 and -6 and tumor necrosis factor-α was statistically higher in the OLETF rats. Finally, histological analysis showed more severe fibrosis and loss of lamellar pattern in AF tissues of OLETF rats. Conclusion Our results suggest that diabetes mellitus is associated with increased apoptosis and expression of matrix degrading enzymes and inflammatory cytokines in AF cells. This results in more severe fibrosis and loss of lamellar pattern of AF, which leads to intervertebral disc degeneration. Strict DM control might be important to delay or prevent early intervertebral disc degeneration in patients with DM.

Expression and significance of telomerase in the nucleus pulposus tissues of degenerative lumbar discs.

The pathogenesis of lumbar disc degeneration is extremely complex, and the expression and role of telomerase in degenerative lumbar disc tissues remains unclear. The aim of the present study was to detect telomerase expression in nucleus pulposus tissues of degenerative lumbar discs and to explore the correlation between telomerase expression and other factors typical of disc degeneration. A total of 8 patients with degenerative nucleus pulposus were included as the experimental group and compared with 8 control patients without evident lumbar disc degeneration. The expression of telomerase in nucleus pulposus tissues was detected by immunohistochemical staining. ELISA was performed to determine the differential expression of telomerase, type II collagen and chondroitin sulfate between the two groups. In addition, a correlation analysis was performed to form associations between these factors. Finally, 5 cases in the experimental group and 5 in the control group were involved in the analysis. Immunohistochemistry results showed that telomerase expression in the experimental group was significantly lower compared to the control group and the percentage in the unit field of view showed significant differences between the two groups (P<0.05). Similarly, the ELISA test results showed lower expression levels of telomerase, type II collagen and chondroitin sulfate in the experimental group when compared with the control group (P<0.05). The correlation analysis revealed that telomerase was positively correlated with type II collagen and chondroitin sulfate (correlation coefficients, 0.673 and 0.528, respectively; P<0.01). In conclusion, telomerase is involved in the degeneration process of nucleus pulposus tissue in lumbar discs and has a positive correlation with other factors typically associated with degeneration.

Damage accumulation location under cyclic loading in the lumbar disc shifts from inner annulus lamellae to peripheral annulus with increasing disc degeneration

It is difficult to study the breakdown of lumbar disc tissue over several years of exposure to bending and lifting by experimental methods. In our earlier published study we have shown how a finite element model of a healthy lumbar motion segment was used to predict the damage accumulation location and number of cyclic to failure under different loading conditions. The aim of the current study was to extend the continuum damage mechanics formulation to the degenerated discs and investigate the initiation and progression of mechanical damage. Healthy disc model was modified to represent degenerative discs (Thompson grade III and IV) by incorporating both geometrical and biochemical changes due to degeneration. Analyses predicted decrease in the number of cycles to failure with increasing severity of disc degeneration. The study showed that the damage initiated at the posterior inner annulus adjacent to the endplates and propagated outwards towards its periphery in healthy and grade III degenerated discs. The damage accumulated preferentially in the posterior region of the annulus. However in grade IV degenerated disc damage initiated at the posterior outer periphery of the annulus and propagated circumferentially. The finite element model predictions were consistent with the infrequent occurrence of rim lesions at early age but a much higher incidence in severely degenerated discs.

The correlation between microvessel pathological changes of the endplate and degeneration of the intervertebral disc in diabetic rats

In this study, the pathological microvessel changes to the endplate and the degeneration of the intervertebral disc of diabetic rats were examined in order to identify the possible mechanism by which diabetes mellitus (DM) induces degeneration of the intervertebral disc. A total of 30 Sprague-Dawley rats were randomly divided into two groups. DM was induced in one of the groups by streptozotocin (STZ) administration. The rats were sacrificed 4, 8 and 12 weeks later. Five rats from each group were sacrificed at each time interval and lumbar disc and endplate tissue were obtained from each rat. The histological changes, collagen expression, microvessel density (MVD) and apoptosis of the disc were investigated by different methods. The expression of collagen I in the diabetic DM group was higher compared to the control group at the three time points (P<0.01), in contrast to the expression of collagen II. The factor VIII-related antigen (FVIII RAg) was expressed in the control and DM groups, while its expression was relatively low in the DM group. The MVD of the DM group was smaller compared to that of the control group at the three time points (P<0.01). The apoptotic index (AI) in the diabetic group was significantly higher compared to that of the control group at the three time points (P<0.01). A negative correlation was observed between the MVD of the endplates and the notochordal cell AI in the two groups. Compared to the control group, the endplate MVD decreased and the cavity became smaller or disappeared in the diabetic rats. In conclusion, there was a negative correlation between MVD and degenerative changes of the intervertebral disc in diabetic rats.

Dorsoventral organization of sensory nerves in the lumbar spine as indicated by double labeling of dorsal root ganglion neurons

BackgroundReferred pain due to lumbar disc disorders can be analyzed using the stereoscopic structure of the peripheral sensory nervous system. The rostrocaudal structure has been clarified. The dorsoventral structure of the lumbar spine would be useful for mapping areas of pain perception in spinal disorders. MethodsThe neurotracer 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) was applied to the lateral portion of the L5/6 intervertebral disc in rats to examine the dorsoventral organization of the sensory nervous system in the lumbar spine and related tissues. Fluorogold (FG) was applied to reference sites located at the spinous process of the L5 vertebra, the L5/6 facet joint, the psoas muscle at the L5 level, or the rectus abdominis muscle at the pubic symphysis. FG was also applied to the lateral portion of the disc (DiI application site) at L5 or at the L5 level as controls for the double labeling. Labeled neurons were counted in dorsal root ganglia (DRGs) from L1 through L4. ResultsThe proportion of neurons double-labeled with DiI and FG in the total number of DiI-labeled and FG-labeled neurons was 32.9% in the control group; 0% in the spinous process, 0.6% in the facet joint, 2.3% in the psoas muscle, and 0.1% in the rectus abdominis muscle. DRG neurons with dichotomizing afferent fibers were most prevalent (2.3%) between the lateral disc and the psoas muscle at the groin; they were rare or absent between the disc and other reference sites. ConclusionsDorsoventral organization of the primary sensory system in the lumbar body trunk was suggested from the proportion of DRG neurons with dichotomizing afferent fibers innervating the lumbar disc and other tissues. The present findings provide a pathomechanism of groin referred pain in lumbar disc disorders.

The Occurrence and Regional Distribution of DR4 on Herniated Disc Cells

Intervertebral discs surgically obtained from 60 herniated patients and 5 normal individuals were examined to correlate the regional distribution of DR4-receptor and apoptosis. To explore the role of a tumor necrosis factor superfamily member DR4 and the TRAIL/DR4 mediated apoptosis in the human lumbar intervertebral disc. The pathogenesis of lumbar degenerative intervertebral discs remains not completely understood. In herniated lumbar disc tissues, increased apoptosis and higher expression of Fas/Fas ligand and caspase-3 have been reported, suggesting a pivotal role of apoptotic mechanisms in intervertebral disc degeneration. However, it is not clear that apoptosis mediators such as TRAIL and Death Receptor 4 (DR4), which often represent different apoptosis signal pathways, contribute to the apoptosis process during the development of the degenerated intervertebral discs. Apoptosis was determined by poly(ADP-ribose) polymerase (PARP) p85 immunohistochemistry. Expression of DR4 was revealed by immunohistochemistry analysis. Statistical difference among groups was analyzed using one-way ANOVA with LSD post hoc multiple comparisons and the bivariate correlations. Apoptotic cells were detected in the nucleus pulposus and anulus fibrosus of all samples. However, the number of apoptotic cells was significantly higher in the nucleus compared with the anulus. Further, there were significantly more apoptotic cells in the herniated discs compared with the normal discs. Within herniated discs, a remarkably higher percentage of positive staining cells were detected in the uncontained discs than the contained ones. Strong expression of DR4 was detected in all samples of degenerative herniated discs, whereasmuch weaker expression was sporadically identified in normal discs. In addition, the prevalence of apoptosis positively correlated with the severity of disc degeneration. The concomitant increase of DR4 expression in the regions of heavy apoptotic cell aggregation suggests that TRAIL/DR4-mediated pathway may play an important role in the apoptosis in herniated discs.

Analysis of Tissue Distribution of TNF‐α, TNF‐α‐Receptors, and the Activating TNF‐α–Converting Enzyme Suggests Activation of the TNF‐α System in the Aging Intervertebral Disc

We immunohistochemically analyzed the expression and localization of TNF-alpha, its receptors TNF-RI and -RII, and the TNF-alpha-activating enzyme TACE in human autopsy (n=63) and surgical (n=35) lumbar intervertebral disc samples. In parallel, the TNF-alpha-mRNA was quantified by reverse transcriptase-polymerase chain reaction (RT-PCR). All samples were morphometrically evaluated for the proportion of positively labeled cells in the different anatomical regions of the disc. We detected a significant and comparable expression of all four parameters beginning in young adult age (c. 18 years) and being most extensive in the nucleus pulposus. This level was slightly reduced in older age discs. The annulus fibrosus contained significantly less labeled cells. In accord, the number of TNF-alpha-transcripts was elevated in most cases with immunohistochemical TNF-alpha expression. We provide clear evidence that TNF-alpha is expressed in discs of increasing age, which correlates with histomorphological signs of disc degeneration. In consequence, TNF-alpha seems to be activated (by the converting enzyme TACE) and biologically active through its receptors in human lumbar disc tissue.

Analysis and prevalence of inflammatory cells in subtypes of lumbar disc herniations under cyclooxygenase-2 inhibitor therapy

Objective: The aim of the present study was to analyse inflammatory cells in lumbar disc tissue under cyclooxygenase-2 (COX-2) inhibitor therapy, to detect their prevalence in different subtypes of lumbar disc herniations and to assess the influence of inflammatory reactions in herniated disc tissue on postoperative outcome.Methods: In this prospective study, intervertebral disc specimens were obtained from 50 patients. All the patients were given COX-2 inhibitor therapy (Rofecoxib) 25 mg/day for 10 days before surgery. The herniated disc specimens were routinely fixed in a 10% buffered formaline solution and paraffin-embedded; 5 &mum thin sections were stained with monoclonal antibodies CD-68 for macrophages, CD-45 RO for T cells and CD-20 for B cells. The specimens were microscopically examined and classified by two independent examiners in a blinded manner.Results: CD-68 macrophages were evident in herniated lumbar disc tissue in 40% of the cases, but abundant inflammation was observed in only 18% of the cases. Macrophages were significantly common in sequesters with a prevalence of 80%, and no macrophages were observed in the protrusions. We obtained the best outcome scores in the patients with a sequestrated lumbar disc herniation.Conclusion: Although most of the specimens from all the patients showed no significant inflammatory reaction, maybe due to COX-2 inhibitor therapy, they all needed surgery with no relief of the symptoms despite conservative therapy. Nerve root compression still seems to be the leading pathomechanism.

Mitochondrial Involvement in Fas-Mediated Apoptosis of Human Lumbar Disc Cells

Two main pathways of Fas-mediated apoptosis have been identified: the Type-I (death-inducing signaling complex) pathway and the Type-II (mitochondrial) pathway. While apoptotic cell death has been implicated in lumbar degenerative disc disease, we are not aware of any studies in which surgically removed discs from live humans have been examined to determine which of the two pathways is involved in the apoptosis of disc cells. As an initial step in the development of therapies to inhibit inappropriate or premature apoptosis of disc cells, our objective was to determine which pathway is involved. We examined thirty-two samples of herniated lumbar disc tissue with use of immunohistochemical staining and Western blot analysis to determine the presence of several proteins, including caspase-8 (associated with the Type-I pathway); BID (BH3 interacting domain death agonist), cytochrome-c, and caspase-9 (associated with the Type-II pathway); and caspase-3 (an executioner of apoptosis). The TUNEL (terminal deoxynucleotidyl transferase [TDT]-mediated dUTP nick end labeling) assay was performed to confirm the occurrence of apoptosis of the disc cells. The proteins associated with the Type-II pathway (BID, cytochrome-c, and caspase-9) stained positively in all samples. Although the protein associated with the Type-I pathway (caspase-8) was not detected on immunohistochemical analysis, a small amount of caspase-8 was detected on Western blot analysis. However, the expression of Type-II proteins was still higher than the expression of caspase-8 on Western blot analysis. The expression of caspase-3 was identified in all samples with immunohistochemical and Western blot analysis. TUNEL-positive disc cells were identified in all samples. The results of the present study suggest that human disc cells are Type-II cells, which undergo apoptotic cell death through mitochondrial involvement.