Development of a novel animal model of lumbar vertebral endplate lesion by intervertebral disk injection of monosodium iodoacetate in rats.

PURPOSE: Vertebral endplate lesions (EPLs) caused by severe disk degeneration are associated with low back pain. However, its pathophysiology remains unclear. In this study, we aimed to develop a vertebral EPL rat model mimicking severe intervertebral disk (IVD) degeneration by injecting monosodium iodoacetate (MIA) into the IVDs and evaluating it by assessing pain-related behavior, micro-computed tomography (CT) findings, and histological changes. METHODS: MIA was injected into the L4-5 and L5-6 IVDs of Sprague-Dawley rats. Their behavior was examined by measuring the total distance traveled and the total number of rearing in an open square arena. Bone alterations and volume around the vertebral endplate were assessed using micro-CT. Safranin-O staining, immunohistochemistry, and tartrate-resistant acid phosphatase (TRAP) staining were performed for histological assessment. RESULTS: The total distance and number of rearing times in the open field were significantly reduced in a time-dependent manner. Micro-CT revealed intervertebral osteophytes and irregularities in the endplates at 12 weeks. The bone volume/tissue volume (BV/TV) around the endplates significantly increased from 6 weeks onward. Safranin-O staining revealed severe degeneration of IVDs and endplate disorders in a dose- and time-dependent manner. Calcitonin gene-related peptide-positive nerve fibers significantly increased from 6 weeks onward. However, the number of osteoclasts decreased over time. CONCLUSION: Our rat EPL model showed progressive morphological vertebral endplate changes in a time- and concentration-dependent manner, similar to the degenerative changes in human IVDs. This model can be used as an animal model of severe IVD degeneration to better understand the pathophysiology of EPL.

Tungsten accumulates in the intervertebral disc and vertebrae stimulating disc degeneration and upregulating markers of inflammation and pain.

Tungsten is incorporated in many industrial goods, military applications and medical devices due to its ability to impart flexibility, strength and conductance to materials. Emerging evidence has questioned the safety of tungsten exposure as studies have demonstrated it can promote tumour formation, induce pulmonary disease and alter immune function. Although tungsten is excreted from the body it can accumulate in certain organs such as the brain, colon, liver, kidneys, spleen and bones, where most of the bioaccumulation occurs. Whether prolonged tungsten exposure leads to accumulation in other tissues is unknown. The present study demonstrated that mice exposed to 15 ppm sodium tungstate for 4 weeks in their drinking water showed comparable accumulation in both the bony vertebrae and intervertebral discs (IVDs). Lumbar IVD height was significantly reduced in tungsten-exposed mice and accompanied by decreased proteoglycan content and increased fibrosis. In addition to catabolic enzymes, tungsten also increased the expression of the inflammatory cytokines IL-1ß and tumour necrosis factor (TNF)-α as well as the neurotrophic factors nerve growth factor (NGF) and brain-derived nerve factor (BDNF) in IVD cells. Tungsten significantly increased the presence of nociceptive neurons at the endplates of IVDs as observed by the expression of calcitonin gene-related peptide (CGRP) and anti-protein gene product 9.5 (PGP9.5) in endplate vessels. The present study provided evidence that tungsten may enhance disc degeneration and fibrosis as well as increase the expression of markers for pain. Therefore, tungsten toxicity may play a role in disc degeneration disease.

Melatonin protects vertebral endplate chondrocytes against apoptosis and calcification via the Sirt1-autophagy pathway.

Melatonin is reportedly associated with intervertebral disc degeneration (IDD). Endplate cartilage is vitally important to intervertebral discs in physiological and pathological conditions. However, the effects and mechanism of melatonin on endplate chondrocytes (EPCs) are still unclear. Herein, we studied the effects of melatonin on EPC apoptosis and calcification and elucidated the underlying mechanism. Our study revealed that melatonin treatment decreases the incidence of apoptosis and inhibits EPC calcification in a dose-dependent manner. We also found that melatonin upregulates Sirt1 expression and activity and promotes autophagy in EPCs. Autophagy inhibition by 3-methyladenine reversed the protective effect of melatonin on apoptosis and calcification, while the Sirt1 inhibitor EX-527 suppressed melatonin-induced autophagy and the protective effects of melatonin against apoptosis and calcification, indicating that the beneficial effects of melatonin in EPCs are mediated through the Sirt1-autophagy pathway. Furthermore, melatonin may ameliorate IDD in vivo in rats. Collectively, this study revealed that melatonin reduces EPC apoptosis and calcification and that the underlying mechanism may be related to Sirt1-autophagy pathway regulation, which may help us better understand the association between melatonin and IDD.

Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration.

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert-butyl hydroperoxide (TBHP)-treated nucleus pulposus cells of SD rats in vitro as well as in a puncture-induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen-II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts-5. Additionally, autophagy blockade using 3-MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine-treated rats had an accentuated T2-weighted signal and a diminished histological degenerative grade than vehicle-treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.

Time-Course Investigation of Intervertebral Disc Degeneration Induced by Different Sizes of Needle Punctures in Rat Tail Disc.

BACKGROUND This study aimed to determine the best size needle to use in inducing IVDD and to find the proper time point of disc degeneration suitable for further biologic treatment study. MATERIAL AND METHODS First, rat tail level 5/6, 7/8, and 9/10 discs were punctured by 18G, 21G, or 25G needles. Then, degeneration was assessed by radiography, MRI, and histological evaluation at 2, 4, and 6 weeks after puncture. Later, real-time reverse transcriptase (RT-PCR) was used to examine mRNA expressions of aggrecan, collagen type II, hypoxia-inducible factor-1a (HIF-1a), glucose transporter1 (GLUT-1), and vascular endothelial growth factor (VEGF). RESULTS Significant differences were identified in almost all parameters compared with the control group in the 18G and 21G group at almost all time points. To assess the effect of different needle sizes on DHI, we used magnetic resonance imaging (MRI), grade, and mRNA expression. We found significant differences between different groups, except for DHI between the 21G group and 25G group and MRI grade between the 18G and 21G group at the 2-week time point. In assessing the effect of different needle sizes on HE staining score and toluidine blue staining grade, statistical differences were observed at some time points. The effects of time on all parameters were significant at almost all time points in all groups. CONCLUSIONS The middle-size needle (21G) performed better in inducing disc degeneration. The 2-week time point may be better for use in further experimental studies.

Intervertebral disc degenerative changes after intradiscal injection of TNF-α in a porcine model.

PURPOSE: To investigate whether exogenous tumor necrosis factor-α (TNF-α) will initiate a degenerative process in intervertebral disc in vivo. METHODS: Exogenous TNF-α in dosages of 50 and 100 ng in 50 µL Dulbecco's Modified Essential Medium (DMEM) was injected into porcine lumbar discs; a third disc was injected only with 50 µL DMEM as a control. Magnetic resonance imaging (MRI) yielding T1- and T2-weighted images, T2-mapping, and post-contrast T1 images was performed and histology was studied as well. RESULTS: After 3 months, a significant decrease in T2 value calculated from T2-mapping MRI was observed in the annulus and nucleus of both groups injected with TNF-α along with a slight decrease in disc height and nucleus volumes in comparison to the control discs. No obvious visual differences among the groups were observed in the normal T1- and T2-weighted MRI images. Post-contrast T1 MRI showed increased annulus enhancement in both TNF-α-injected groups compared to the control discs, while no enhancement difference was observed in the nucleus. Histological analysis showed degenerative changes with annulus fissure, cell cluster, nucleus matrix loss, vascularization and interleukin-1ß expression in the outer annulus of both TNF-α-injected discs, while no degenerative changes were observed in the control discs. CONCLUSIONS: Intradiscal injection of exogenous TNF-α caused early stage disc degeneration in a porcine model. It may thus support the hypothesis of exogenic TNF-α being an important early pathogenetic factor in disc degeneration.

In vivo experimental intervertebral disc degeneration induced by bleomycin in the rhesus monkey.

BACKGROUND: Recently, biological therapies for early intervention of degenerative disc disease have been introduced and developed; however, a functional animal model that mimics slowly progressive disc degeneration of humans does not exist. The objective of this study was to establish a slowly progressive and reproducible intervertebral disc (IVD) degeneration model. METHODS: The subchondral bone adjacent to the lumbar IVDs (L3/4 and L5/6) of ten rhesus monkeys was randomly injected with 4 ml bleomycin solution (1.5 mg/ml), or 4 ml phosphate buffer saline (PBS) per segment as control, respectively. The degenerative process was investigated by using radiography and T1ρ MR imaging at 1, 3, 6, 9, 12 and 15 months postoperatively. Histological scoring, Sulfated Glycosaminoglycans (GAGs) analysis and real-time PCR were performed at 15 months. The correlation between histological score, GAGs and T1ρ values were also analyzed. RESULTS: The results showed that the mean T1ρ values of nucleus pulposus (NP) and annulus fibrosus (AF) in the bleomycin group significantly decreased after 3 and 6 months respectively, followed by slowly decrease until at 15 months. At 15 months, the histological scores was significantly higher, and the GAGs of NP was significantly lower in the bleomycin group, compared with the control group (P<0.05). The results of real-time PCR revealed a significant increase in matrix metalloprotease (MMP)-3, A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, tumor necrosis factor α, interleukin-1ß, interleukin-6 expressions, transforming growth factor (TGF-ß1) and marked reduction in aggrecan, type II collagen, von willebrand factor (vWF) expressions at the mRNA levels in the bleomycin group. Spearman correlation analysis showed a strong positive correlation between GAGs and T1ρ values of NP (r =0.740, P<0.01), and a significant inverse correlation between histological score and T1ρ values of NP and AF (r=-0.761, r=-0.729, respectively, P<0.01). CONCLUSIONS: Injection of bleomycin into the subchondral bone adjacent to the lumbar IVDs of rhesus monkeys can results in mild, slowly progressive disc degeneration, which mimics the onset of human disc degeneration. T1ρ MR imaging is an effective and noninvasive technique for assessment of early stage disc degeneration.

[A novel rabbit disc degeneration model induced by fibronectin fragment].

OBJECTIVE: To establish a novel and useful rabbit model of lumbar disc degeneration using microinjection of fibronectin fragment (Fn-f). METHODS: Thirty-two New Zealand white rabbits underwent injection of N-terminal 30 kDa Fn-f (experimental group) or phosphate buffered saline (PBS) (control group) into the central region of L1-2, L2-3, L3-4, L4-5 discs using a 32-gauge microsyringe. Two rabbits (blank group) with no treatments were sacrificed to examine the proteoglycan synthesis of neucleus pulposus (NP) using (35)S-sulfate incorporation assay. At the 4-, 8-, 12-, and 16-week time points, the discs were examined histologically, radiographically, and with proteoglycan synthesis. RESULTS: Histology demonstrated a progressive loss of the cell numbers in NP and architecture destruction in NP and anulus fibrosus (AF) in Fn-f-injected discs over the 16-week study period. The NP regions in Fn-f-injected discs shrinked distinctly after the 4-week time point, and were not discernible with the inner AF by the 16-week time point. Protoglycan synthesis in Fn-f-injected discs decreased progressively (F = 263.241, P = 0.000). At each time point, the Fn-f-injected discs showed significantly decreased proteoglycan synthesis compared with controls (t = -27.010 - -2.833, P < 0.05). The DHI% of the Fn-f-injected discs at the 4-, 8-, 12-, and 16-week time points were 96.5% ± 1.7%, 85.6% ± 3.8%, 77.2% ± 3.5% and 65.5% ± 5.6%, respectively. Comparing with the DHI% of PBS-injected discs (97.4% ± 1.2%), the Fn-f-injected discs exihibited no significant differences in disc heights at the 4-week time point (P > 0.05), but significant decreases in disc heights at the 8-, 12-, and 16-week time points (t = -21.225 - -10.795, P < 0.01). Apparent anterior osteophytes formed at the 12-week time point and enlarged remarkablely by the 16-week time point in the experimental spines. CONCLUSIONS: Fn-f can induce a progressively degenerative process in rabbit discs which is ethical, cost-effective, reproducible, and consistent with the spontaneous degeneration in human. And it seem to be a novel and useful model for the study of disc degeneration at the molecular level.

Establishment of an Animal Model of Disk Degeneration by Intradiskal Injection of Monosodium Iodoacetate.

BACKGROUND: Disk degeneration (DD) stands for the most common cause of low back pain. The establishment of an animal model plays an intrinsic role in the clarification of the physiopathology of DD. The purpose of this study is to select an optimal dose of monosodium iodoacetate (MIA) that may generate a reliable model of DD. METHODS: Thirty-four rats were used in this study. The disks (Co7/8, Co8/9, and Co 9/10) received 1 shot of intradiskal injection of 0.02 mg, 0.1 mg, and 0.5 mg of MIA solution, respectively. Half of the rats were euthanized 3 weeks after MIA injection, and the other half 6 weeks after injection. RESULTS: Magnetic resonance imaging evaluation showed that the mean T2-weighted signal intensity at 6 weeks decreased significantly in the 0.1 and 0.5 mg groups. The disk height of the control group was significantly higher than those of the 0.1 mg and 0.5 mg groups. Histologic and macroscopic results revealed time-and-dose-depending degeneration in the disks that received MIA. Additionally, MIA produced cell death in the nucleus pulposus cells with an elevated percentage. The injected disk with 0.1 mg MIA demonstrated a progressive degeneration, the disk injected with 0.5 mg MIA induced DD acutely 3 weeks post MIA injection, while the dose of 0.02 mg of MIA did not show much degeneration. CONCLUSIONS: We concluded that 0.1 mg MIA is the most suitable dose to establish a model of DD, which enabled us to replicate the onset, progression, and outcome of diverse histopathologies of DD in the clinic.

Spine degeneration in a murine model of chronic human tobacco smokers.

OBJECTIVE: To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS: Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS: Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS: These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

Development of a novel model of intervertebral disc degeneration by the intradiscal application of monosodium iodoacetate (MIA) in rat.

BACKGROUND CONTEXT: Low back pain is one of the most common musculoskeletal disorders. Although, the pathology of intervertebral disc (IVD) degeneration has been modeled using various biological methods, these models are inadequate for simulating similar pathologic states in humans. PURPOSE: This study investigated whether monosodium iodoacetate (MIA) injection into the IVD of rats could generate a reliable model of IVD degeneration. STUDY DESIGN/SETTINGS: In vivo animal study. METHODS: MIA was injected into two-disc spaces (L4-5 and L5-6) of Sprague-Dawley rats. Their behaviors were examined by measuring weight load shifts from hind to forefoot, rearing, and von Frey tests. We examined the inhibition of pain behavior through intraperitoneal morphine injection and measured cyclooxygenase-2 (COX-2) and transcription factor nuclear factor-kappa B (NF-κB) levels in the IVD and dorsal root ganglion (DRG) by Western blot. Bone alterations were assessed by microfocus computed tomography (micro-CT), and IVD and/or cartilage changes were evaluated by hematoxylin and eosin and safranin-O staining and inducible nitric oxide synthase (iNOS) immunohistochemistry. The other authors declare no conflicts of interest. This project funded by the Memorial Fund and the National Research Foundation of Korea (NRF). RESULTS: We observed increased weight load shifts to the forefoot and decreased rearing. Morphine-injected rats showed reduced pain. NF-κB and COX-2 expression increased in the IVD and left and/or right DRG. Micro-CT analyses suggested progressive bone deformation. Histologic examination showed decreased IVD width and nucleus pulposus area. Cartilaginous changes indicated epiphyseal growth plate loss. Finally, iNOS expression was increased in the subchondral endplate. CONCLUSIONS: These results suggest that low back pain (LBP) models can be developed by MIA injection into the IVDs of rats and that an animal model is useful for exploring degenerative alterations in the affected discs. Therefore, MIA injection may be a useful model for the study of changes in the IVD to elucidate the mechanisms underlying clinical symptoms, such as LBP, in patients with IVD degeneration. CLINICAL SIGNIFICANCE: This model in which MIA was injected into the disc better represented the human histologic and behavioral characteristics than the existing puncture model.

Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.

The present work describes a novel bovine disc organ culture system with long-term maintenance of cell viability, in which degenerative changes can be induced as a prelude to studying repair. Discs were isolated with three different techniques: without endplates (NEP), with bony endplates (BEP) and with intact cartilage endplates (CEP). Swelling, deformation, and cell viability were evaluated in unloaded cultures. Degeneration was induced by a single trypsin injection into the center of the disc and the effect on cell viability and matrix degradation was followed. Trypsin-treated discs were exposed to TGFß to evaluate the potential to study repair in this system. NEP isolated discs showed >75% maintained cell viability for up to 10 days but were severely deformed, BEP discs on the other hand maintained morphology but failed to retain cell viability having only 27% viable cells after 10 days. In CEP discs, both cell viability and morphology were maintained for at least 4 weeks where >75% of the cells were still viable. To mimic proteoglycan loss during disc degeneration, a single trypsin injection was administered to the center of the disc. This resulted in 60% loss of aggrecan, after 7 days, without affecting cell viability. When TGFß was injected to validate that the system can be used to study a repair response following injection of a bio-active substance, proteoglycan synthesis nearly doubled compared to baseline synthesis. Trypsin-treated bovine CEP discs therefore provide a model system for studying repair of the degenerate disc, as morphology, cell viability and responsiveness to bio-active substances were maintained.

Intradiscal injection of monosodium iodoacetate induces intervertebral disc degeneration in an experimental rabbit model.

BACKGROUND: Establishing an optimal animal model for intervertebral disc (IVD) degeneration is essential for developing new IVD therapies. The intra-articular injection of monosodium iodoacetate (MIA), which is commonly used in animal models of osteoarthritis, induces cartilage degeneration and progressive arthritis in a dose- and time-dependent manner. The purpose of this study was to determine the effect of MIA injections into rabbit IVDs on the progression of IVD degeneration evaluated by radiographic, micro-computerized tomography (micro-CT), magnetic resonance imaging (MRI), and histological analyses. METHODS: In total, 24 New Zealand White (NZW) rabbits were used in this study. Under general anesthesia, lumbar discs from L1-L2 to L4-L5 had a posterolateral percutaneous injection of MIA in contrast agent (CA) (L1-L2: CA only; L2-L3: MIA 0.01 mg; L3-L4: 0.1 mg; L4-L5: 1.0 mg; L5-L6: non-injection (NI) control). Disc height was radiographically monitored biweekly until 12 weeks after injection. Six rabbits were sacrificed at 2, 4, 8, and 12 weeks post-injection and processed for micro-CT, MRI (T2-mapping), and histological analyses. Three-dimensional (3D) disc height in five anatomical zones was evaluated by 3D reconstruction of micro-CT data. RESULTS: Disc height of MIA-injected discs (L2-L3 to L4-L5) gradually decreased time-dependently (P < 0.0001). The disc height of MIA 0.01 mg-injected discs was significantly higher than those of MIA 0.1 and 1.0 mg-injected discs (P < 0.01, respectively). 3D micro-CT analysis showed the dose- and time-dependent decrease of 3D disc height of MIA-injected discs predominantly in the posterior annulus fibrosus (AF) zone. MRI T2 values of MIA 0.1 and 1.0 mg-injected discs were significantly decreased compared to those of CA and/or NI controls (P < 0.05). Histological analyses showed progressive time- and dose-degenerative changes in the discs injected with MIA (P < 0.01). MIA induced cell death in the rabbit nucleus pulposus with a high percentage, while the percentage of cell clones was low. CONCLUSIONS: The results of this study showed, for the first time, that the intradiscal injection of MIA induced degenerative changes of rabbit IVDs in a time- and dose-dependent manner. This study suggests that MIA injection into rabbit IVDs could be used as an animal model of IVD degeneration for developing future treatments.

Inhibition of both endplate nutritional pathways results in intervertebral disc degeneration in a goat model.

BACKGROUND: The vertebral endplate route was demonstrated to be the main pathway for nutrition to the intervertebral disc. However, it is still a controversial issue on whether the blocking of the endplate nutritional pathway could result in intervertebral disc degeneration (IDD) in animal models. The aim was therefore to investigate the effect of the inhibition of both endplate nutritional pathways by bone cement injection on the IDD in a goat model. METHODS: Two lumbar intervertebral discs (L2-3 and L3-4) in eight 24-month-old goats were blocked in both endplate nutritional pathways by cement injection, and the other two lumbar intervertebral discs (L1-2 and L4-5) remained intact as normal controls. Effective blocking area percentage in nucleus pulposus (NP) was calculated, and X-rays, magnetic resonance imaging (MRI), and histology studies were performed at 4, 12, 24, and 48 weeks after operation. RESULTS: The mean effective blocking area percentage was 60.7 ± 5.3%. Imaging examinations at the time of 48 weeks after blocking the endplate nutritional pathways showed obvious IDD, with larger disc height reduction and higher degrees of disc degeneration grading compared with the normal controls. Histological examinations including HE, Masson's trichrome, Sirius Red, and proteoglycan stainings also confirmed the degenerative changes of the blocked discs. CONCLUSIONS: The endplate nutritional route could be inhibited by blocking both endplate pathways with cement injection in a goat model. The severe inhibition in the endplate nutritional pathways may result in IDD.

Magnetic resonance imaging evaluation of the effects of cigarette and maras powder (smokeless tobacco) on lumbar disc degeneration.

OBJECTIVE: The main aim of this study was to investigate and compare the effects of the use of Maras powder and cigarettes on lumbar disc degeneration. PAIENTS AND METHODS: This study included 87 adult patients who presented at our hospital with a complaint of mechanical low back pain and underwent lumbar magnetic resonance imaging (MRI). Patients meeting the inclusion criteria were divided into three groups. Group 1 included those who smoked Maras powder but not cigarettes, group 2 was comprised of those who smoked cigarettes but did not use Maras powder, and group 3 was comprised of those who did not use tobacco (no cigarettes or Maras powder) (control group). Lumbar disc degeneration was rated according to the Pfirrmann classification. Hematological parameters were obtained from all three groups. RESULTS: Degeneration levels in group 1 were significant when compared to the other groups at all disc levels. Maras powder contributed to intervertebral disc degeneration and this effect increased gradually towards the distal area. The neutrophil to lymphocyte ratio was significantly higher in group 1 than in the other two groups, and was positively correlated with the duration of use of Maras powder (r = 0.689, p ≤ 0.001). CONCLUSION: Although more prevalent in the Maras powder group, lumbar disc degeneration increased significantly with tobacco usage. The results of our study show that Maras powder, commonly used regionally, is likely to cause more spinal disc degeneration than cigarettes.

Effects of Intradiscal Injection of Local Anesthetics on Intervertebral Disc Degeneration in Rabbit Degenerated Intervertebral Disc.

Analgesic discoblock is widely used for the diagnosis or treatment of discogenic low back pain by injecting local anesthetics. The purpose of this study was to investigate the deleterious effects of local anesthetics on degenerated rabbit intervertebral disks (IVDs) using an organotypic culture model and in vivo long-term follow-up model. To induce IVD degeneration, a rabbit annular puncture model was used. For the organotypic culture model, degenerated IVDs were harvested 1 month after the initial annular puncture and cultured for 3 or 7 days after intradiscal injection of local anesthetics (1% lidocaine and 0.5% bupivacaine). To perform in vivo analysis, local anesthetics were injected into degenerated IVDs, and IVDs were prepared for histological analysis after 6 or 12 months. In the organotypic model, terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nucleus pulposus (NP) cells were significantly increased in the bupivacaine group compared with the other groups. In the in vivo study, the number of NP cells was significantly decreased in the saline and local anesthetics groups compared with the untreated control and puncture-only groups. However, there was no significant difference among the saline, lidocaine, and bupivacaine groups. In addition, histological analysis showed no significant difference of IVD degeneration among the puncture-only, saline, lidocaine, and bupivacaine groups. Although bupivacaine induced apoptotic NP cell death in the organotypic culture model, in vivo observations did not show any definitive proof to suggest that local anesthetics were capable of promoting degeneration in the degenerated IVD, except for pressurized injection-induced damage. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1963-1971, 2019.

High glucose promotes annulus fibrosus cell apoptosis through activating the JNK and p38 MAPK pathways.

Diabetes mellitus (DM) is an important risk factor of intervertebral disc degeneration. A high glucose niche-mediated disc cell apoptosis is an implicate causative factor for the spine degenerative diseases related with DM. However, the effects of a high glucose niche on disc annulus fibrosus (AF) cell apoptosis and the potential signaling transduction pathway is unclear. The present study is to investigate the effects of high glucose on disc AF cell apoptosis and the role of two potential signaling pathways in this process. Rat AF cells were cultured in baseline medium or medium with different concentrations (0.1 and 0.2 M) of glucose for 3 days. Flow cytometry was used to assess the degree of apoptosis. Activity of caspase 3/9 was evaluated by chemical kit. Expression of pro-apoptotic and anti-apoptotic molecules was analyzed by real-time polymerase chain reaction and Western blot. In addition, activity of the C-Jun NH2-terminal kinases (JNK) pathway and p38 mitogen-activated protein kinase (MAPK) pathway was evaluated by Western blot. Compared with the control group, high glucose culture increased cell apoptosis ratio and caspase-3/9 activity, up-regulated expression of bax, caspase-3, cleaved caspase-3 and cleaved PARP, and down-regulated expression of bcl-2 in a glucose concentration-dependent manner. Additionally, high glucose culture increased expression of the p-JNK and p-p38 MAPK in a concentration-dependent manner. Further results showed that inhibition of the JNK or p38 MAPK pathway attenuated the effects of high glucose on AF cell apoptosis. Together, high glucose promoted disc AF cell apoptosis through regulating the JNK pathway and p38 MAPK pathway in a glucose concentration-dependent manner.

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 in vitro environment.

Overexpression of miR-140-5p inhibits lipopolysaccharide-induced human intervertebral disc inflammation and degeneration by downregulating toll-like receptor 4.

Toll-like receptor 4 (TLR4) families are receptors for ligands that initiate extracellular or intracellular signaling, such as lipopolysaccharides (LPS). It has been reported that TLR4 activation resulted in the upregulation of a coordinated set of proinflammatory mediators and inhibition of matrix expression in the intervertebral disc (IVD). miR-140-5p (miR-140) is reported to participate in cellular anti-inflammatory processes and target TLR4. In the present study, we investigated the relationship between TLR4 and miR-140 in IVD degeneration. The expression of TLR4, interleukin (IL)-6, IL-I, L-1ß and tumor necrosis factor (TNF)-α was higher, in high-grade IVD degeneration tissues than in low-grade tissues. In contrast, the expression of miR-140, aggrecan and collagen type II was lower in high-grade IVD degeneration tissues than in low-grade IVD degeneration tissues. LPS stimulation resulted in significant increases in TLR4 expression and decreases in miR-140 expression in nucleus pulposus (NP) cells and TLR4 was identified as a target of miR-140 by dual-luciferase reporter assay. The overexpression of miR-140 inhibited the upregulation of the expression of TLR4, TNF-α, IL-1ß and IL-6 inflammation cytokines, and the activation of NF-κB and reversed the downregulation of the expression of aggrecan and collagen type II induced by LPS stimulation. In conclusion, the present study may lead to a greater understanding of IVD degeneration and provide new insights into the treatment of this disease.

Enhancement of Lumbar Fusion and Alleviation of Adjacent Segment Disc Degeneration by Intermittent PTH(1-34) in Ovariectomized Rats.

Osteoporosis, which is prevalent in postmenopausal or aged populations, is thought to be a contributing factor to adjacent segment disc degeneration (ASDD), and the incidence and extent of ASDD may be augmented by osteopenia. Parathyroid hormone (PTH) (1-34) has already been shown to be beneficial in osteoporosis, lumbar fusion and matrix homeostasis of intervertebral discs. However, whether PTH(1-34) has a reversing or retarding effect on ASDD in osteopenia has not been confirmed. In the present study, we evaluated the effects of intermittent PTH(1-34) on ASDD in an ovariectomized (OVX) rat model. One hundred 3-month-old female Sprague-Dawley rats underwent L4 -L5 posterolateral lumbar fusion (PLF) with spinous-process wire fixation 4 weeks after OVX surgery. Control groups were established accordingly. PTH(1-34) was intermittently administered immediately after PLF surgery and lasted for 8 weeks using the following groups (n = 20) (V = vehicle): Sham+V, OVX+V, Sham+PLF+V, OVX+PLF+V, OVX+PLF+PTH. The fused segments showed clear evidence of eliminated motion on the fusion-segment based on manual palpation. Greater new bone formation in histology was observed in PTH-treated animals compared to the control group. The extent of ASDD was significantly increased by ovariotomy. Intermittent PTH(1-34) significantly alleviated ASDD by preserving disc height, microvessel density, relative area of vascular buds, endplate thickness and the relative area of endplate calcification. Moreover, protein expression results showed that PTH(1-34) not only inhibited matrix degradation by decreasing MMP-13, ADAMTS-4 and Col-I, but also promote matrix synthesis by increasing Col-II and Aggrecan. In conclusion, PTH(1-34), which effectively improves lumbar fusion and alleviates ASDD in ovariectomized rats, may be a potential candidate to ameliorate the prognosis of lumbar fusion in osteopenia.