Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-ß, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

Inhibition of cytochrome P450c17 reduces spinal astrocyte activation in a mouse model of neuropathic pain via regulation of p38 MAPK phosphorylation.

We have recently demonstrated that the neurosteroid-metabolizing enzyme, cytochrome P450c17 is increased in spinal astrocytes contributing to the development of mechanical allodynia in chronic constriction injury (CCI)-induced neuropathic mice. However, the mechanisms by which spinal P450c17 modulates pathological changes in astrocytes remain unclear. In this study we investigated whether P450c17 modulates astrocyte activation and whether this process is mediated by spinal p38 mitogen-activated protein kinase phosphorylation ultimately leading to the development of mechanical allodynia in CCI mice. Sciatic nerve injury induced a significant increase in glial fibrillary acidic protein (GFAP) expression in the superficial dorsal horn (SDH, laminae I-II) and nucleus proprius (NP, laminae III-IV) regions of the spinal cord dorsal horn. Repeated daily (from days 0-3 post-surgery) intrathecal administration of the P450c17 inhibitor, ketoconazole (10 nmol) significantly inhibited the CCI-induced increase in GFAP-immunoreactivity, but had no effect on the CCI-induced increase in Iba-1-immunoreactivity. In addition, intrathecal administration of ketoconazole significantly inhibited the CCI-induced increase in p38 phosphorylation, while the levels of ERK and JNK phosphorylation remained unchanged. The CCI-induced development of mechanical allodynia was attenuated by administration of either ketoconazole (10 nmol) or the p38 MAPK inhibitor, SB203580 (5 nmol). Administration of a sub-effective dose of SB203580 (0.5 nmol) potentiated the pharmacological effect of ketoconazole (1 nmol) on spinal GFAP-immunostaining, as well as, the development of mechanical allodynia following CCI. Collectively these data suggest that spinal P450c17 activates astrocytes via p38 phosphorylation, ultimately leading to the development of mechanical allodynia in a model of peripheral neuropathy.

Matrix metalloproteinase promotes elastic fiber degradation in ligamentum flavum degeneration.

Ligamentum flavum (LF) hypertrophy in lumbar spinal canal stenosis (LSCS) is characterized by a loss of elastic fibers and fibrosis. Chronic inflammation is thought to be responsible for the histological change but the mechanism underlying elastic fiber degradation remains unclear. Given that matrix metalloproteinase (MMP)-2 and -9 have elastolytic activity and are partly regulated by inflammatory cytokines such as interleukin (IL)-6, in this study, we investigated whether MMPs mediate LF degeneration using 52 LF samples obtained during lumbar surgery, including 31 LSCS and 21 control specimens. We confirmed by histological analysis that the LSCS samples exhibited severe degenerative changes compared with the controls. We found that MMP-2 was upregulated in LF tissue from patients with LSCS at the mRNA and protein levels, whereas MMP-9 expression did not differ between the two groups. The MMP-2 level was positively correlated with LF thickness and negatively correlated with the area occupied by elastic fibers. IL-6 mRNA expression was also increased in LF tissue from patients with LSCS and positively correlated with that of MMP-2. Signal transducer and activator of transcription (STAT)3, a component of the IL-6 signaling pathway, was activated in hypertrophied LF tissues. Our in vitro experiments using fibroblasts from LF tissue revealed that IL-6 increased MMP-2 expression, secretion, and activation via induction of STAT3 signaling, and this effect was reversed by STAT3 inhibitor treatment. Moreover, elastin degradation was promoted by IL-6 stimulation in LF fibroblast culture medium. These results indicate that MMP-2 induction by IL-6/STAT3 signaling in LF fibroblasts can degrade elastic fibers, leading to LF degeneration in LSCS.

Effects of Ovariectomy in an hSOD1-G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS).

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by progressive muscular dystrophy and paralysis; most ALS patients die from respiratory failure within 3 to 5 years, and there is currently no effective treatment. Some studies have indicated sex differences in the incidence of ALS, and evidence suggests a neuroprotective role for estrogen. MATERIAL AND METHODS We used human Cu/Zn superoxide dismutase (hSOD1-G93A) transgenic mice to determine the effects of ovariotomy on the onset of disease and behavior; we also used Western blotting to measure the expression of aromatase and estrogen receptors, as well as the inflammatory cytokines and apoptosis markers, in the lumbar spinal cord to determine the mechanism of estrogen-mediated neuroprotection. RESULTS Ovariectomy advanced the onset of disease, down-regulated aromatase and estrogen receptor alpha (ER-a) expression, and inhibited expression of the anti-inflammatory factors arginase-1 and the anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) in the lumbar spinal cord of hSOD1-G93A transgenic mice. CONCLUSIONS Ovariectomy resulted in earlier disease onset and attenuated the anti-inflammatory and anti-apoptotic actions of estrogen in hSOD1-G93A transgenic mice. Therefore, estrogen may play an important role in protecting spinal cord motor neurons.

Correlation Between Expression of High Temperature Requirement Serine Protease A1 (HtrA1) in Nucleus Pulposus and T2 Value of Magnetic Resonance Imaging.

BACKGROUND Degrading enzymes play an important role in the process of disc degeneration. The objective of this study was to investigate the correlation between the expression of high temperature requirement serine protease A1 (HtrA1) in the nucleus pulposus and the T2 value of the nucleus pulposus region in magnetic resonance imaging (MRI). MATERIAL AND METHODS Thirty-six patients who had undergone surgical excision of the nucleus pulposus were examined by MRI before surgery. Pfirrmann grading of the target intervertebral disc was performed according to the sagittal T2-weighted imaging, and the T2 value of the target nucleus pulposus was measured according to the median sagittal T2 mapping. The correlation between the Pfirrmann grade and the T2 value was analyzed. The expression of HtrA1 in the nucleus pulposus was analyzed by RT-PCR and Western blot. The correlation between the expression of HtrA1 and the T2 value was analyzed. RESULTS The T2 value of the nucleus pulposus region was 33.11-167.91 ms, with an average of 86.64±38.73 ms. According to Spearman correlation analysis, there was a rank correlation between T2 value and Pfirrmann grade (P<0.0001), and the correlation coefficient (rs)=-0.93617. There was a linear correlation between the mRNA level of HtrA1 and T2 value in nucleus pulposus tissues (a=3.88, b=-0.019, F=112.63, P<0.0001), normalized regression coefficient=-0.88. There was a linear correlation between the expression level of HtrA1 protein and the T2 value in the nucleus pulposus tissues (a=3.30, b=-0.016, F=93.15, P<0.0001) and normalized regression coefficient=-0.86. CONCLUSIONS The expression of HtrA1 was strongly related to the T2 value, suggesting that HtrA1 plays an important role in the pathological process of intervertebral disc degeneration.

Eliglustat maintains long-term clinical stability in patients with Gaucher disease type 1 stabilized on enzyme therapy.

In the phase 3 Study of Eliglustat Tartrate (Genz-112638) in Patients With Gaucher Disease Who Have Reached Therapeutic Goals With Enzyme Replacement Therapy (ENCORE), at 1 year, eliglustat was noninferior to imiglucerase enzyme therapy in maintaining stable platelet counts, hemoglobin concentrations, and spleen and liver volumes. After this primary analysis period, patients entered a long-term extension phase in which all received eliglustat. Duration on eliglustat ranged from 2 to 5 years, depending on timing of enrollment (which spanned 2 years), treatment group to which patients were randomized, and whether they lived in the United States when commercial eliglustat became available. Here we report long-term safety and efficacy of eliglustat for 157 patients who received eliglustat in the ENCORE trial; data are available for 46 patients who received eliglustat for 4 years. Mean hemoglobin concentration, platelet count, and spleen and liver volumes remained stable for up to 4 years. Year to year, all 4 measures remained collectively stable (composite end point relative to baseline values) in ≥85% of patients as well as individually in ≥92%. Mean bone mineral density z scores (lumbar spine and femur) remained stable and were maintained in the healthy reference range throughout. Eliglustat was well tolerated over 4 years; 4 (2.5%) patients withdrew because of adverse events that were considered related to the study drug. No new or long-term safety concerns were identified. Clinical stability assessed by composite and individual measures was maintained in adults with Gaucher disease type 1 treated with eliglustat who remained in the ENCORE trial for up to 4 years. This trial was registered at www.clinicaltrials.gov as #NCT00943111.

Involvement of Spinal Angiotensin II System in Streptozotocin-Induced Diabetic Neuropathic Pain in Mice.

Renin-angiotensin system (RAS) activity increases under hyperglycemic states, and is thought to be involved in diabetic complications. We previously demonstrated that angiotensin (Ang) II, a main bioactive component of the RAS, might act as a neurotransmitter and/or neuromodulator in the transmission of nociceptive information in the spinal cord. Here, we examined whether the spinal Ang II system is responsible for diabetic neuropathic pain induced by streptozotocin (STZ). Tactile allodynia was observed concurrently with an increase in blood glucose levels the day after mice received STZ (200 mg/kg, i.v.) injections. Tactile allodynia on day 14 was dose-dependently inhibited by intrathecal administration of losartan, an Ang II type 1 (AT1) receptor antagonist, but not by PD123319, an AT2 receptor antagonist. In the lumbar dorsal spinal cord, the expression of Ang II, Ang converting enzyme (ACE), and phospho-p38 mitogen-activated protein kinase (MAPK) were all significantly increased on day 14 after STZ injection compared with vehicle-treated controls, whereas no differences were observed among AT1 receptors or angiotensinogen levels. Moreover, the increase in phospho-p38 MAPK was significantly inhibited by intrathecal administration of losartan. These results indicate that the expression of spinal ACE increased in STZ-induced diabetic mice, which in turn led to an increase in Ang II levels and tactile allodynia. This increase in spinal Ang II was accompanied by the phosphorylation of p38 MAPK, which was shown to be mediated by AT1 receptors.

The NLRP3/Caspase-1/Interleukin-1ß Axis Is Active in Human Lumbar Cartilaginous Endplate Degeneration.

BACKGROUND: Modic changes are the MRI signal changes of degenerative lumbar vertebral endplate and which lead to or accelerate intervertebral disc degeneration. NLRP3, caspase-1, and interleukin-1ß (IL-1ß) play a pivotal role in the pathogenesis of many inflammatory diseases, such as osteoarthritis. However, the roles of IL-1ß and its activators caspase-1 and NLRP3 are unclear in the degenerative endplate. QUESTIONS/PURPOSES: We asked: (1) What are the degenerative changes of the histologic features and chondrogenic markers' gene expressions between the cartilaginous endplates of patients with Modic changes and trauma (control)? (2) How does the NLRP3/caspase-1/IL-1ß axis in the cartilaginous endplates of patients with Modic changes compare with control (trauma) specimens? METHODS: Surgical specimens of cartilaginous endplates were divided into Modic changes (n = 56) and the trauma control (n = 16) groups. Hematoxylin and eosin and safranin O staining of cartilaginous endplate tissues were done to evaluate the extracellular matrix. Reverse transcription-polymerase chain reaction was performed on these tissues to investigate mRNA expression of type II collagen (Col II), SOX-9, matrix metalloproteinase-3, and a disintegrin like and metalloproteinase thrombospondin type I motifs-5. NLRP3, caspase-1, and IL-1ß were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: Hematoxylin and eosin and safranin O staining showed the extracellular matrix degraded in the cartilaginous endplates of patients with Modic changes but not in the control cartilaginous endplates. Chondrogenic Col II (p = 0.024) and SOX9 (p = 0.053) were downregulated in the Modic changes group compared with the control group. In contrast to the control group, the transcriptional levels of NLRP3 (p < 0.001), caspase-1 (p = 0.054), and IL-1ß (p = 0.001) were all upregulated in the Modic changes group. CONCLUSIONS: The expression of NLRP3, caspase-1, and IL-1ß was upregulated in the patients with low back pain and Modic changes on MRI compared with patients with vertebral burst fracture without degenerative changes on MRI. The data suggest the NLRP3/caspase-1/IL-1ß axis may be implicated in lumbar cartilaginous endplate degeneration. CLINICAL RELEVANCE: The NLRP3/caspase-1/IL-1ß axis is active in cartilaginous endplates of patients with Modic changes and inflammatory cascades can exacerbate the cartilaginous endplate degeneration which may act as a trigger for intervertebral disc degeneration and low back pain. If these findings can be confirmed by others, we hope that new and effective therapy could be developed directed against this target.

Residual enzymatic activity as a prognostic factor in patients with Gaucher disease type 1: correlation with Zimran and GAUSS-I index and the severity of bone disease.

BACKGROUND: Gaucher disease (GD) is an autosomal recessive disorder produced by mutations in the glucocerebrosidase gene (GBA), causing storage of glucosylceramide in reticuloendothelial cells in multiple organs. Traditionally, the prediction of the phenotype based on the genotype has been reported to be limited. SUBJECTS AND METHODS: We investigated the correlation between the enzymatic residual activity (ERA) and the phenotype at diagnosis of the disease in 45 GD Spanish patients (44 with type I and 1 with type III GD). The genotype involved two of the following previously expressed proteins: c.517A > C (T134P), 1%; c.721G > A (G202R), 17%; c.1090G > T (G325W), 13.9%; c.1208G > A (S364N), 4.1%; c.1226A > G (N370S), 17.8%; c.1246G > A (G377S), 17.6%; c.1289C > T (P391L), 8.5%; c.1448T > C (L444P), 3%; and c.1504C > T (R463C), 24.5%. Recombinant alleles, deletion of 55 bp in exon 9 and 84GG mutation were considered as mutations with no residual enzymatic activity. RESULTS: The ERA showed a statistically significant correlation with chitotriosidase (P < 0.001), age (P < 0.001), spleen size (P < 0.001), 'Zimran's Severity Score Index' (P < 0.01) and the 'Gaucher Disease Severity Score Index-Type I' (P < 0.0001) at diagnosis of the disorder. Previous to any medical intervention, a comparison between the ERA and bone involvement, demonstrated a statistically significant relationship (P < 0.01) between the two variables. CONCLUSIONS: This study data allowed us to define a new criterion for prognostic assessment of the disease at diagnosis, called Protein Severity Index, which expresses the theoretical severity of the genotype presented by patients, according to the corresponding ERA.

Intrathecal injection of phosphodiesterase 4B-specific siRNA attenuates neuropathic pain in rats with L5 spinal nerve ligation.

Phosphodiesterase 4 (PDE4) is an adenosine cyclic 3,5-monophosphate-specific degradative enzyme, which is closely associated with the inflammatory response. Among its four subtypes (A-D), it remains unclear which one exerts suppressive effects on inflammation and reduces neuropathic pain. The present study aimed to examine the modulation of neuroinflammation by PDE4 subtypes in the spinal cord of a rat model of L5 spinal nerve ligation (SNL)-induced neuropathic pain. The expression levels of PDE4A-D were measured in the lumbar spinal cords of naïve rats. The rats were then divided into seven groups: The sham group (sham surgery + saline), the saline group (SNL + saline), the vehicle group (SNL + Lipofectamine® RNAiMAX), the mismatch small interfering (si)RNA group (SNL + mismatch siRNA), the PDE4A-siRNA group (SNL + PDE4A-siRNA), the PDE4B-siRNA group (SNL + PDE4B-siRNA) and the PDE4D-siRNA group (SNL + PDE4D-siRNA). In order to determine behavioral changes, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded. The mRNA and protein expression levels of PDE4s were also detected. Furthermore, the association between behavioral changes and individual subtypes of PDE4 were studied following intrathecal administration of PDE4A, B and D-specific siRNA. The expression levels of protein kinases, including phosphorylated-extracellular signal-regulated kinases (p-ERK), and inflammatory cytokines were measured, in order to explore the underlying mechanisms. Subtypes A, B and D, but not C, were detected in the naïve rats. After SNL, both MWT and TWL were reduced. The mRNA and protein expression levels of PDE4A, B and D were significantly upregulated after 2, 4, 6 and 8 days of SNL. Subtype-specific siRNA significantly suppressed the elevated expression levels; however, only rats treated with PDE4B siRNA exhibited improved MWT and TWL. Further analysis of the PDE4B siRNA-treated rats demonstrated that 8 days after SNL, the intensity of p-ERK was reduced, the expression levels of CD11b and glial fibrillary acidic protein GFAP were reduced, as well as the expression levels of proinflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1ß and IL-6. These results suggested that selective inhibition of PDE4B may relieve neuropathic pain, potentially via the suppression of glial activation and the release of cytokines in the spinal cord.

Effect of acupotomy on nitric oxide synthase and beta-endorphin in third lumbar vertebrae transverse process syndrome model rats.

OBJECTIVE: To explore the long-term effects and pain relief mechanism of acupotomy by observing changes in nitric oxide synthase (NOS) and beta-endorphin (beta-EP) in the hypothalamus, spinal cord, and peripheral blood of rats with third lumbar vertebrae (L3) transverse process syndrome. METHODS: Twenty-eight SD rats were randomly assigned to normal, model, electroacupuncture (EA), and acupotomy group. The last three groups were put through an operation to emulate L3 transverse process syndrome. Fourteen days after the simulation operation, EA and acupotomy treatments were applied to the respective groups. Fifty-six days after the simulation operation, biochemistry tests and enzyme-linked immunosorbent assay were used to measure NOS and beta-EP in the hypothalamus, spinal cord, and peripheral blood. RESULTS: Rats with the simulation operation showed significantly higher levels of NOS and beta-EP in the hypothalamus, spinal cord, and peripheral blood than those in the normal group. The EA and acupotomy groups had significantly lower levels of NOS and beta-EP than those in the model group. There was no statistical difference between the EA and acupotomy groups. CONCLUSION: EA and acupotomy treatments significantly lowered NOS and beta-EP levels in the hypothalamus, spinal cord, and peripheral blood and alleviated L3 transverse process syndrome.

Phenytoin and sodium valproate but not levetiracetam induce bone alterations in female mice.

Adverse effects on the bone are amongst the potentially adverse clinical consequences with antiepileptic drugs (AEDs). This study compared the effects of 3 AEDs (phenytoin (PHT), sodium valproate (SVP), and levetiracetam (LTM)) on the bones of a Swiss strain of albino female mice. Drugs were administered daily for 4 months at doses that produced plasma concentrations corresponding to the clinically relevant therapeutic ranges. PHT and SVP (but not LTM) significantly lowered the bone mineral density (BMD) of lumbar vertebrae (L2-L4) as evaluated by dual-energy X-ray absorptiometry (DEXA) scan. The findings were supported by histopathology of vertebral (lumbar) bone and analysis of bone turnover markers. While both PHT and SVP reduced alkaline phosphatase (ALP) and hydroxyproline (HxP) in lumbar vertebrae, and elevated tartarate-resistant acid phosphatase (TRAP) and urinary excretion of calcium, LTM did not affect any of these markers of bone turnover, indicating that the drug might be a safer option in female epileptic patients prone to bone changes.

Developing a mechanical and chemical model of degeneration in young bovine lumbar intervertebral disks and reversing loss in mechanical function.

STUDY DESIGN: A mechanical and chemical model of intervertebral disk (IVD) degeneration was developed by examining the enzymatic degradation of the nucleus pulposus (NP), and a gelatin-based restoration study was performed. OBJECTIVE: It was hypothesized that forced enzymatic degradation of the NP will mimic natural degeneration through the loss of disk height and that an injection of a gelatin solution will restore mechanical function. SUMMARY OF BACKGROUND DATA: Collagen and proteoglycans are essential for normal NP function. Their chemical destruction, combined with light mechanical loading, will mimic degeneration. Previous studies have determined that collagenase and matrix metalloproteinase-3 are directly implicated in IVD degradation; therefore, these enzymes were used in this model. MATERIALS AND METHODS: On the basis of preliminary testing, 0.5% collagenase, 1% collagenase, and 0.0025% metalloproteinase-3 in phosphate-buffered saline (PBS) were injected directly into the NP of various motion segments from a young bovine lumbar spine and subjected to light cyclic loading. To restore disk height and mechanical function, 20% gelatin in PBS at 70°C was injected into a degraded disk and subjected to the same loading conditions after an allotted hardening time. RESULTS: Mechanical testing showed statistically significant changes in disk height between control segments, 1% collagenase, and 0.5% collagenase. 0.5% collagenase had the most accurate appearance and loading pattern of degeneration upon disk transection postloading. A trend in restoration of disk function, given by the lessened loss of disk height upon loading, was observed with injection of gelatin after degradation with 0.5% collagenase. CONCLUSIONS: This study demonstrated the potential to create a degenerative model using enzymatic degradation of the NP and the possibility to restore function with an injectable therapy. Although gelatin is not a clinically viable option, it provides preliminary data for other injectable IVD therapies.

Overexpression of BDNF increases excitability of the lumbar spinal network and leads to robust early locomotor recovery in completely spinalized rats.

Strategies to induce recovery from lesions of the spinal cord have not fully resulted in clinical applications. This is a consequence of a number of impediments that axons encounter when trying to regrow beyond the lesion site, and that intraspinal rearrangements are subjected to. In the present study we evaluated (1) the possibility to improve locomotor recovery after complete transection of the spinal cord by means of an adeno-associated (AAV) viral vector expressing the neurotrophin brain-derived neurotrophic factor (BDNF) in lumbar spinal neurons caudal to the lesion site and (2) how the spinal cord transection and BDNF treatment affected neurotransmission in the segments caudal to the lesion site. BDNF overexpression resulted in clear increases in expression levels of molecules involved in glutamatergic (VGluT2) and GABAergic (GABA, GAD65, GAD67) neurotransmission in parallel with a reduction of the potassium-chloride co-transporter (KCC2) which contributes to an inhibitory neurotransmission. BDNF treated animals showed significant improvements in assisted locomotor performance, and performed locomotor movements with body weight support and plantar foot placement on a moving treadmill. These positive effects of BDNF local overexpression were detectable as early as two weeks after spinal cord transection and viral vector application and lasted for at least 7 weeks. Gradually increasing frequencies of clonic movements at the end of the experiment attenuated the quality of treadmill walking. These data indicate that BDNF has the potential to enhance the functionality of isolated lumbar circuits, but also that BDNF levels have to be tightly controlled to prevent hyperexcitability.

Effects of vibration in forced posture on biochemical bone metabolism indices, and morphometric and mechanical properties of the lumbar vertebra.

Epidemiological studies have shown a relatively strong association between occupational lower back pain (LBP) and long-term exposure to vibration. However, there is limited knowledge of the impact of vibration and sedentariness on bone metabolism of the lumbar vertebra and the mechanism of bone-derived LBP. The aim of this study was to investigate the effects of vibration in forced posture (a seated posture) on biochemical bone metabolism indices, and morphometric and mechanical properties of the lumbar vertebra, and provide a scientific theoretical basis for the mechanism of bone-derived LBP, serum levels of Ca(2+), (HPO4)(2-), tartrate-resistant acid phosphatase (TRAP), bone-specific alkaline phosphatase (BALP), and bone gla protein (BGP),the pathological changes and biomechanics of lumbar vertebra of New Zealand white rabbits were studied. The results demonstrate that both forced posture and vibration can cause pathological changes to the lumbar vertebra, which can result in bone-derived LBP, and vibration combined with a seated posture could cause further damage to bone metabolism. Serological changes can be used as early markers for clinical diagnosis of bone-derived LBP.

Expression of hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinases 1, 3, and 9 in hypertrophied ligamentum flavum.

STUDY DESIGN: Immunohistological study. OBJECTIVE: To elucidate the role of matrix metalloproteinases (MMPs), hypoxia-inducible factor-1α (HIF), and vascular endothelial growth factor (VEGF) in the hypertrophied ligamentum flavum (LF) obtained from patients with lumbar spinal stenosis (LSS). SUMMARY OF BACKGROUND DATA: The most common spinal disorder in the elderly is LSS, which results in part from LF hypertrophy. Although prior histologic and immunochemical studies have been performed in this area, the pathophysiology of loss of elasticity and hypertrophy is not completely understood. METHODS: LF samples of 38 patients with LSS were harvested during spinal decompression. Twelve LF samples obtained from patients with disk herniation and no visible degeneration on preoperative magnetic resonance imaging were obtained as controls. Samples were dehydrated and paraffin embedded. For immunohistochemical determination of VEGF, HIF, and MMPs 1, 3, and 9 expression, slices were stained with VEGF, HIF, and MMP antibody dilution. Neovessel density and number of elastic fibers were counted after Masson-Goldner staining. LF hypertrophy and cross-sectional area (CSA) were measured on T1-weighted magnetic resonance imaging. RESULTS: MMPs 1, 3, 9 and VEGF expression were significantly increased in the hypertrophy group (P<0.05). HIF expression was negative in both groups. Vessel density was increased in the hypertrophy group, although this was not statistically significant. The number of elastic fibres was significantly higher in the control group. In the hypertrophy group, LF thickness was significantly increased, whereas CSA was significantly decreased. There was a statistical correlation between LF thickness, CSA, MMP, and VEGF expression in the hypertrophy group (P<0.05). CONCLUSIONS: LF hypertrophy is accompanied by increased MMPs 1, 3, 9 and VEGF expression. Neovessel density is increased in hypertrophied LF. HIF is not expressed in hypertrophied LF.

Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons.

BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. RESULTS: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and phospho-CREB, and reverses cystitis-induced increases in micturition frequency. CONCLUSIONS: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.

Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain.

BACKGROUND: Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK) family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs) limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. RESULTS: We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs) as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide). Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro-inflammatory cytokines and chemokines and the phosphorylated form of p38. CONCLUSIONS: Our results indicate that MKP-1, the natural regulator of p-p38, mediates resolution of the spinal cord pro-inflammatory milieu induced by peripheral nerve injury, resulting in prevention of chronic mechanical hypersensitivity. We propose that MKP-1 is a potential therapeutic target for pain treatment or prevention.

Abundance of calpain and aggrecan-cleavage products of calpain in degenerated human intervertebral discs.

OBJECTIVE: To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs). DESIGN: Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and µ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA. RESULTS: Discs at early stages of degeneration expressed low levels of m- and µ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, µ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous. CONCLUSIONS: The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage.

The role of matrix metalloproteinase 9 in intervertebral disc degeneration.

The present study investigates the histological alterations and expression of matrix metalloproteinase 9 (MMP-9) in disc specimens of 43 patients who underwent surgery for lumbar disc herniation. The immunostaining for MMP-9 was evaluated semi-quantitatively. Histologic degeneration was scored between 0 and 12 depending on the degree of chondrocyte proliferation and presence of tears and clefts, granular changes and mucous degeneration. Herniation was graded as grade 1 (protrusion), grade 2 (extrusion) or grade 3 (sequestration) on MRI. Although there was no significant statistical difference between the histologic degeneration score and age, degenerative changes were more pronounced in higher grade of herniation (p<0.0001). MMP-9 expression was related to histologic degenerative score in all age groups (p=0.0065). MMP-9 was also related to herniation grade in patients younger than 30 years of age (p=0.0037). No significant association was found between MMP-9 expression and herniation grade in patients who were 30-60 years or over 60 years of age.