Inhibiting IκB kinase-ß downregulates inflammatory cytokines in injured discs and neuropeptides in dorsal root ganglia innervating injured discs in rats.

STUDY DESIGN: Quantitative and immunohistological analysis of the efficacy of an IκB kinase-ß (IKKß) inhibitor in an injured intervertebral disc (IVD) model. OBJECTIVE: To elucidate the efficacy of an IKKß inhibitor on inflammatory cytokine levels in injured IVDs or on neuropeptide levels in the dorsal root ganglia (DRG) neurons innervating injured IVDs in rats. SUMMARY OF BACKGROUND DATA: Multiple studies have suggested that upregulation of inflammatory cytokines in damaged IVDs causes discogenic low back pain. The efficacy of blocking individual inflammatory cytokines is limited; however, inflammatory cytokine stimuli often require IKKß to activate nuclear factor-k B. METHODS: Sprague-Dawley rats were divided into 3 groups: sham, saline (disc-injury plus saline), and IKKß (disc-injury plus anti-IKKß). To induce injury, IVDs were repeatedly punctured.Experiment 1: Four, 7, and 14 days postinjury, coccygeal (Co) 5/6, Co6/7, and Co7/8 IVDs were resected and tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 levels were quantified by enzyme-linked immunosorbent assay. Experiment 2: The neurotracer Fluoro-Gold was injected into injured L5-L6 IVDs and uninjured sham group IVDs to detect DRG neurons. One week postsurgery, L1-L6 DRGs were immunolabeled with the neuropeptide calcitonin gene-related peptide. The proportions of Fluoro-Gold-labeled calcitonin gene-related peptide-immunoreactive DRG neurons were assessed. RESULTS: Experiment 1: IVD levels of tumor necrosis factor-α (through 2 wk), IL-1ß (at 4 d), and IL-6 (at 4 d) were significantly higher in the saline group than in the sham group, and significantly lower in the IKKß group than in the saline group (P < 0.05). Experiment 2: The percentage of calcitonin gene-related peptide-immunoreactive Fluoro-Gold-labeled DRG neurons was significantly higher in the saline group than in the sham group, and significantly lower in the IKKß group than in the saline group (P < 0.05). CONCLUSION: Injury-induced upregulation of inflammatory cytokines within IVDs and increased levels of neuropeptides within DRG neurons can be suppressed by inhibiting IKKß. LEVEL OF EVIDENCE: N/A.

IκB kinase ß inhibitor downregulates pain-related neuropeptide production in the sensory neurons innervating injured lumbar intervertebral discs in the dorsal root ganglia of rats.

BACKGROUND CONTEXT: Nuclear factor-κB (NF-κB) is an essential gene transcriptional regulator of inflammatory cytokines, and it plays important roles in numerous conditions, including inflammatory and neuropathic pain, especially when discogenic pain is involved. Phosphorylation of IκB protein through IκB kinase (IKK) is the first step in the activation of NF-κB activation and the upregulation of NF-κB-responsive genes. PURPOSE: To investigate whether IKK inhibition alters the properties of pain-related neuropeptides in the rat lumbar degenerative intervertebral disc (IVD) model. STUDY DESIGN: Retrograde neurotracing and immunofluorescent investigation of pain-related neuropeptide (calcitonin gene-related peptide [CGRP]) in the sensory innervation of injured lumbar IVD in rat dorsal root ganglia (DRGs). METHODS: Forty female Sprague-Dawley rats were equally divided into four groups: naive, sham, and two agent-treated groups (vehicle [saline] group and anti-IKKß [IMD-0560, IKKß inhibitor] group). The L5-L6 IVDs of the agent-treated rats were exposed and injured by repeated punctures. The retrograde neurotracer Fluoro-Gold (FG) and corresponding treatment agents were intradiscally applied. In the sham group, FG alone was applied onto uninjured IVDs. One week later, L1-L3 DRGs were harvested and immunolabeled for CGRP as a pain marker. The proportions of FG-labeled CGRP-immunoreactive (-ir) DRG neurons were assessed. RESULTS: Fluoro-Gold-labeled DRG neurons were almost equally prevalent at each DRG level. The proportions of FG-labeled CGRP-ir DRG neurons in the two agent-treated groups were significantly increased in comparison with those in the naive and the sham groups (p<.05) and were significantly decreased in the anti-IKKß group in comparison with that in the vehicle group (p<.05). CONCLUSIONS: The neuropeptide CGRP as a pain marker was upregulated in DRG neurons innervating the injured IVDs, and intradiscal inhibition of IKKß significantly suppressed CGRP production in the DRG neurons innervating the rat IVD, suggesting the possible analgesic effect of IKKß inhibition in discogenic pain.

Radial shock waves effectively introduced NF-kappa B decoy into rat achilles tendon cells in vitro.

The purpose of this study was to test if radial shock waves could enhance the introduction of nuclear factor-kappa B (NF-kappaB) decoy oligodeoxynucleotides, which is reported to markedly inhibit NF-kappaB activation and suppress pro-inflammatory cytokine gene expression, using rat Achilles tendon cells. In the presence of NF-kappaB decoy labeled with or without fluorescein isothiocyanate (FITC) in culture media, radial shock waves were applied to the tendon cells in variable conditions and cultivated for 24 h. The transfection rate was assessed by counting FITC-positive cells, and IL-1-induced NF-kappaB activation in the cells was assessed. Radial shock waves significantly enhanced introduction of NF-kappaB decoy-FITC into the tendon cells. IL-1-induced NF-kappaB activation was significantly inhibited by pretreatment of the cells with NF-kappaB decoy combined with radial shock wave exposure. The present study demonstrated the effectiveness of radial shock waves on introduction of NF-kappaB decoy into tendon cells. Radial shock wave treatment combined with local NF-kappaB decoy administration could be a novel therapeutic strategy for chronic tendinopathy.

Nuclear factor-kappa B decoy suppresses nerve injury and improves mechanical allodynia and thermal hyperalgesia in a rat lumbar disc herniation model.

Nuclear factor-kappa B (NF-kappaB) is a gene transcriptional regulator of inflammatory cytokines. We investigated the transduction efficiency of NF-kappaB decoy to dorsal root ganglion (DRG), as well as the decrease in nerve injury, mechanical allodynia, and thermal hyperalgesia in a rat lumbar disc herniation model. Forty rats were used in this study. NF-kappaB decoy-fluorescein isothiocyanate (FITC) was injected intrathecally at the L5 level in five rats, and its transduction efficiency into DRG measured. In another 30 rats, mechanical pressure was placed on the DRG at the L5 level and nucleus pulposus harvested from the rat coccygeal disc was transplanted on the DRG. Rats were classified into three groups of ten animals each: a herniation + decoy group, a herniation + oligo group, and a herniation only group. For behavioral testing, mechanical allodynia and thermal hyperalgesia were evaluated. In 15 of the herniation rats, their left L5 DRGs were resected, and the expression of activating transcription factor 3 (ATF-3) and calcitonin gene-related peptide (CGRP) was evaluated immunohistochemically compared to five controls. The total transduction efficiency of NF-kappaB decoy-FITC in DRG neurons was 10.8% in vivo. The expression of CGRP and ATF-3 was significantly lower in the herniation + decoy group than in the other herniation groups. Mechanical allodynia and thermal hyperalgesia were significantly suppressed in the herniation + decoy group. NF-kappaB decoy was transduced into DRGs in vivo. NF-kappaB decoy may be useful as a target for clarifying the mechanism of sciatica caused by lumbar disc herniation.

Pericellular matrix formation alters the efficiency of intracellular uptake of oligonucleotides in osteosarcoma cells.

One of the crucial roles of tumor extracellular matrix is to act as a barrier to drug delivery. In this study, we analyzed the relationship between the formation of tumor extracellular matrix and the efficiency of intracellular uptake of oligonucleotides in human osteosarcoma cell lines, HOS, and MG-63. Oligonucleotides used in this study were nuclear factor-kappa B (NF-kappaB) decoy, which might be a therapeutic tool for neoplasms. Pericellular matrix formation was examined by particle exclusion assay. Cellular uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy was evaluated by fluorescent microscopy and flow cytometry. Effects of NF-kappaB decoy on cell viability and cell cycle arrest in MG-63 cells were determined by MTT assay and flow cytometry, respectively. MG-63 cells exhibited abundant pericellular matrix with time compared with HOS cells. Uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy decreased in MG-63 cells with time but not in HOS cells in both monolayer and three-dimensional culture using matrigel. However, after enzymatic removal of pericellular matrix, the uptake markedly recovered in MG-63 cells. NF-kappaB decoy inhibited cell proliferation and induced G0/G1 cell cycle arrest in MG-63 cells. These results suggest that abundant pericellular matrix might disturb the uptake of NF-kappaB decoy, and modification of pericellular matrix composition would increase the efficacy of exogenous oligonucleotides treatment for neoplasms.

Injection of nuclear factor-kappa B decoy into the sciatic nerve suppresses mechanical allodynia and thermal hyperalgesia in a rat inflammatory pain model.

STUDY DESIGN: In vitro and in vivo study of a rat inflammatory pain model using nuclear factor-kappa B decoy. OBJECTIVES: To investigate transduction efficiency of nuclear factor-kappa B decoy into dorsal root ganglion, both in vivo and in vitro, and to assess the suppression of inflammatory pain by nuclear factor-kappa B decoy. SUMMARY OF BACKGROUND DATA: Transcription factor nuclear factor-kappa B is reported to play a crucial role in regulating pro-inflammatory cytokine gene expression. We hypothesized that inhibiting nuclear factor-kappa B gene expression with nuclear factor-kappa B decoy may suppress inflammatory pain. METHODS: Nuclear factor-kappa B decoy-fluorescein isothiocyanate (FITC) was induced in explant culture, endoneurally injected into the sciatic nerve, and its transduction efficiency into dorsal root ganglion measured. For behavioral testing, 12 rats received plantar injections of complete Freund's adjuvant and were divided into 3 groups: decoy group, single endoneural injection of 10 microL of nuclear factor-kappa B decoy (n = 4); saline group, single endoneural injection of 10 microL of saline (n = 4); and naïve group, untreated (n = 4). Behavioral testing was performed using von Frey filaments and a Hargreaves device with a heat source. RESULTS: Total transduction efficiency of nuclear factor-kappa B decoy-FITC was 53.6% in vitro and 20.5% in vivo. No statistical differences were observed with respect to types of cell size distributions of all FITC-positive neurons. In behavioral testing, withdrawal latencies or thresholds significantly differed between the decoy group and the saline group from 2 to 14 days after surgery in the mechanical allodynia experiments, and from 2 to 3 days after surgery in the thermal hyperalgesia experiments. CONCLUSIONS: Nuclear factor-kappa B decoy was conveyed and transduced into dorsal root ganglion both in vivo and in vitro. Additionally, nuclear factor-kappa B decoy reduced mechanical allodynia and thermal hyperalgesia in the rat inflammatory pain model, suggesting that inhibition of nuclear factor-kappa B with nuclear factor-kappa B decoy may represent a key mechanism for mediating inflammation or reducing inflammatory pain.

Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage.

OBJECTIVE: To determine the presence of mesenchymal progenitor cells (MPCs) in human articular cartilage. METHODS: Primary cell cultures established from normal and osteoarthritic (OA) human knee articular cartilage were analyzed for the expression of CD105 and CD166, cell surface markers whose coexpression defines mesenchymal stem cells (MSCs) in bone marrow and perichondrium. The potential of cartilage cells to differentiate to adipogenic, osteogenic, and chondrogenic lineages was analyzed after immunomagnetic selection for CD105+/CD166+ cells and was compared with bone marrow-derived MSCs (BM-MSCs). RESULTS: Up to 95% of isolated cartilage cells were CD105+ and approximately 5% were CD166+. The mean +/- SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 +/- 1.93%. Primary cell cultures from OA cartilage contained significantly increased numbers of CD105+/CD166+ cells. Confocal microscopy confirmed the coexpression of both markers in the majority of BM-MSCs and a subpopulation of cartilage cells. Differentiation to adipocytes occurred in cartilage-derived cell cultures, as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105+/CD166+ cells as well as in primary chondrocytes cultured in the presence of osteogenic supplements. Purified cartilage-derived CD105+/CD166+ cells did not express markers of differentiated chondrocytes. However, the cells were capable of chondrocytic differentiation and formed cartilage tissue in micromass pellet cultures. CONCLUSION: These findings indicate that multipotential MPCs are present in adult human articular cartilage and that their frequency is increased in OA cartilage. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.

Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.