Microarray analyses of the dorsal root ganglia support a role for innate neuro-immune pathways in persistent pain in experimental osteoarthritis.

OBJECTIVE: Following destabilization of the medial meniscus (DMM), mice develop experimental osteoarthritis (OA) and associated pain behaviors that are dependent on the stage of disease. We aimed to describe changes in gene expression in knee-innervating dorsal root ganglia (DRG) after surgery, in order to identify molecular pathways associated with three pre-defined pain phenotypes: "post-surgical pain", "early-stage OA pain", and "persistent OA pain". DESIGN: We performed DMM or sham surgery in 10-week old male C57BL/6 mice and harvested L3-L5 DRG 4, 8, and 16 weeks after surgery or from age-matched naïve mice (n = 3/group). RNA was extracted and an Affymetrix Mouse Transcriptome Array 1.0 was performed. Three pain phenotypes were defined: "post-surgical pain" (sham and DMM 4-week vs 14-week old naïve), "early OA pain" (DMM 4-week vs sham 4-week), and "persistent OA pain" (DMM 8- and 16-week vs naïve and sham 8- and 16-week). 'Top hit' genes were defined as P < 0.001. Pathway analysis (Ingenuity Pathway Analysis) was conducted using differentially expressed genes defined as P < 0.05. In addition, we performed qPCR for Ngf and immunohistochemistry for F4/80+ macrophages in the DRG. RESULTS: For each phenotype, top hit genes identified a small number of differentially expressed genes, some of which have been previously associated with pain (7/67 for "post-surgical pain"; 2/14 for "early OA pain"; 8/37 for "persistent OA pain"). Overlap between groups was limited, with 8 genes differentially regulated (P < 0.05) in all three phenotypes. Pathway analysis showed that in the persistent OA pain phase many of the functions of differentially regulated genes are related to immune cell recruitment and activation. Genes previously linked to OA pain (CX3CL1, CCL2, TLR1, and NGF) were upregulated in this phenotype and contributed to activation of the neuroinflammation canonical pathway. In separate sets of mice, we confirmed that Ngf was elevated in the DRG 8 weeks after DMM (P = 0.03), and numbers of F4/80+ macrophages were increased 16 weeks after DMM (P = 0.002 vs Sham). CONCLUSION: These transcriptomics findings support the idea that distinct molecular pathways discriminate early from persistent OA pain. Pathway analysis suggests neuroimmune interactions in the DRG contribute to initiation and maintenance of pain in OA.

Spinal microglial activation in a murine surgical model of knee osteoarthritis.

OBJECTIVE: Microgliosis, the activation of microglial cells, is thought to contribute to synaptic transmission in the dorsal horn and thereby promote chronic pain. The primary aim of this study was to document the temporal profile of dorsal horn microgliosis after destabilization of the medial meniscus (DMM) in wild type (WT) and Adamts5 null mice. Since neuronal fractalkine (CX3CL1) contributes to microgliosis, we assessed its release from dorsal root ganglia (DRG) cultures after DMM. DESIGN: DMM or sham surgery was performed in the right knee of 10-week old male WT, CX3CR1-green fluorescent protein (GFP), or Adamts5 null C57BL/6 mice. Hind paw mechanical allodynia was monitored using von Frey fibers. L4 dorsal horn microgliosis was assessed 4, 8 and 16 weeks after surgery, based on the morphology of Iba1-immunoreactive microglia. DRG cells (L3-L5) were cultured and supernatants collected for fractalkine (FKN) ELISA. RESULTS: In WT mice, numbers of activated microglia were increased 8 and 16 weeks, but not 4 weeks, after DMM but not sham surgery. DRG cultures showed increased basal FKN release at 8 and 16 weeks. Adamts5 null mice did not develop mechanical allodynia up to 16 weeks after DMM. Accordingly, DRG cultures from these mice did not exhibit increased FKN release and dorsal horn microgliosis did not occur. CONCLUSION: DMM surgery leads to late stage dorsal horn microgliosis. The temporal correlation with DRG FKN release suggests it may contribute to microgliosis. Reduced microgliosis in Adamts5 null mice, which are protected from joint damage and associated mechanical allodynia after DMM, suggests that microgliosis is associated with joint damage and accompanying persistent pain.

Therapeutic effects of an anti-ADAMTS-5 antibody on joint damage and mechanical allodynia in a murine model of osteoarthritis.

OBJECTIVE: The primary goal of this study was to test the disease-modifying effect of blocking a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 with a neutralizing monoclonal antibody (mAb) starting 4 weeks after destabilization of the medial meniscus (DMM) in the mouse. We also investigated whether ADAMTS-5 blockade reversed mechanical allodynia and decreased monocyte chemoattractant protein (MCP)-1 production by dorsal root ganglia (DRG) cells. METHODS: Ten-week old male C57BL/6 mice underwent DMM surgery and were either left untreated or treated with anti-ADAMTS-5 mAb or IgG2c isotype control mAb starting 4 weeks after surgery. Knees were collected for histopathology 4 or 12 weeks later. Mechanical allodynia was monitored biweekly in the ipsilateral hind paw through 16 weeks. DRG were collected and cultured 8 weeks after DMM for analysis of MCP-1 production. RESULTS: By 4 weeks after DMM, mild cartilage degeneration was evident in the medial compartment, small osteophytes were present, and subchondral bone sclerosis was established. By 16 weeks after surgery, significant cartilage deterioration was apparent on the medial tibial plateaux and medial femoral condyles, osteophyte size had increased, and subchondral bone sclerosis was maintained. Treatment with ADAMTS-5 mAb from week 4 to 16 after surgery slowed cartilage degeneration and osteophyte growth but did not affect subchondral bone sclerosis. Moreover, ADAMTS-5 blockade resulted in temporary reversal of mechanical allodynia, which correlated with decreased MCP-1 production by cultured DRG cells. CONCLUSIONS: This study suggests therapeutic efficacy of an ADAMTS-5 mAb in the DMM model, when therapy starts early in disease.

Translational development of an ADAMTS-5 antibody for osteoarthritis disease modification.

OBJECTIVE/METHOD: Aggrecanase activity, most notably ADAMTS-5, is implicated in pathogenic cartilage degradation. Selective monoclonal antibodies (mAbs) to both ADAMTS-5 and ADAMTS-4 were generated and in vitro, ex vivo and in vivo systems were utilized to assess target engagement, aggrecanase inhibition and modulation of disease-related endpoints with the intent of selecting a candidate for clinical development in osteoarthritis (OA). RESULTS: Structural mapping predicts the most potent mAbs employ a unique mode of inhibition by cross-linking the catalytic and disintegrin domains. In a surgical mouse model of OA, both ADAMTS-5 and ADAMTS-4-specific mAbs penetrate cartilage following systemic administration, demonstrating access to the anticipated site of action. Structural disease modification and associated alleviation of pain-related behavior were observed with ADAMTS-5 mAb treatment. Treatment of human OA cartilage demonstrated a preferential role for ADAMTS-5 inhibition over ADAMTS-4, as measured by ARGS neoepitope release in explant cultures. ADAMTS-5 mAb activity was most evident in a subset of patient-derived tissues and suppression of ARGS neoepitope release was sustained for weeks after a single treatment in human explants and in cynomolgus monkeys, consistent with high affinity target engagement and slow ADAMTS-5 turnover. CONCLUSION: This data supports a hypothesis set forth from knockout mouse studies that ADAMTS-5 is the major aggrecanase involved in cartilage degradation and provides a link between a biological pathway and pharmacology which translates to human tissues, non-human primate models and points to a target OA patient population. Therefore, a humanized ADAMTS-5-selective monoclonal antibody (GSK2394002) was progressed as a potential OA disease modifying therapeutic.

Inhibition of PC-12 cell differentiation by the immediate early gene fra-1.

The rat pheochromocytoma cell line (PC-12) offers a powerful in vitro model to study the mechanism of growth factor-induced differentiation and proliferation. Within minutes of addition, agents such as nerve growth factor (NGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and dibutyryl cyclic AMP (db cAMP) rapidly activate cellular immediate early genes such as c-fos, c-jun, jun-B, and egr-1. fra-1, a member of the immediate early gene family, follows a distinctly later time course of induction than c-fos, c-jun, jun-B, and egr-1, suggesting that fra-1 may attenuate the action of genes induced earlier. We demonstrate that constitutive expression of fra-1 in PC-12 cells results in pronounced inhibition of NGF-induced differentiation. Transcriptional activation of c-fos, c-jun, jun-B, and egr-1 by NGF, EGF, and db cAMP was down-regulated to a varying extent whereas NGF-induced ornithine decarboxylase (ODC) was not affected. Expression of jun-D was not affected in PC-12 fra-1 cells. Transfection of fos and egr-1 promoter-chloramphenicol acetyl transferase (CAT) plasmid into these stable fra-1-expressing PC-12 cells revealed that repression of fos and egr-1 was exerted at the promoter level. Thus deregulated fra-1 expression may inhibit PC-12 cell differentiation by altering the patterns of immediate early gene expression.

Chemokines and glycoprotein120 produce pain hypersensitivity by directly exciting primary nociceptive neurons.

Human immunodeficiency virus-1 (HIV-1) infection is associated with numerous effects on the nervous system, including pain and peripheral neuropathies. We now demonstrate that cultured rat dorsal root ganglion (DRG) neurons express a wide variety of chemokine receptors, including those that are thought to act as receptors for the HIV-1 coat protein glycoprotein120 (gp120). Chemokines that activate all of the known chemokine receptors increased [Ca(2+)](i) in subsets of cultured DRG cells. Many neurons responded to multiple chemokines and also to bradykinin, ATP, and capsaicin. Immunohistochemical studies demonstrated the expression of the CXCR4 and CCR4 chemokine receptors on populations of DRG neurons that also expressed substance P and the VR1 vanilloid receptor. RT-PCR analysis confirmed the expression of CXCR4, CX3CR1, CCR4, and CCR5 mRNAs in DRG neurons. Chemokines and gp120 produced excitatory effects on DRG neurons and also stimulated the release of substance P. Chemokines and gp120 also produced allodynia after injection into the rat paw. Thus these results provide evidence that chemokines and gp120 may produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons. Chemokine receptor antagonists may be important therapeutic interventions in the pain that is associated with HIV-1 infection and inflammation.

More mysteries of opium reveal'd: 300 years of opiates.

This year is the 300th anniversary of the publication of one of the first books written about opiates and their subjective effects. Since that time the influence of opiates in Western society has grown enormously, as has our knowledge of the mechanisms by which these drugs produce their effects. Wars have been fought over the use of opiates and the economies of several countries depend on their production. In this article, some aspects of the history and effects of opiates on the arts in particular are explored.

HIV-1, chemokines and neurogenesis.

HIV-1 infection of the brain results in a large number of behavioural defecits accompanied by diverse neuropathological signs. However,it is not clear how the virus produces these effects or exactly how the neuropathology and behavioural defecits are related. In this article we discuss the possibility that HIV-1 infection may negatively impact the process of neurogenesis in the adult brain and that this may contribute to HIV-1 related effects on the nervous system. We have previously demonstrated that the development of the dentate gyrus during embryogenesis requires signaling by the chemokine SDF-1 via its receptor CXCR4. We demonstrated that neural progenitor cells that give rise to dentate granule neurons express CXCR4 and other chemokine receptors and migrate into the nascent dentate gyrus along SDF-1 gradients. Animals deficient in CXCR4 receptors exhibit a malformed dentate gyrus in which the migration of neural progenitors is stalled. In the adult, neurogenesis continues in the dentate gyrus. Adult neural progenitor cells existing in the subgranlar zone, that produce granule neurons, express CXCR4 and other chemokine receptors, and granule neurons express SDF-1 suggesting that SDF-1/CXCR4 signaling is also important in adult neurogenesis. Because the cellular receptors for HIV-1 include chemokine receptors such as CXCR4 and CCR5 it is possible that the virus may interfere with SDF-1/CXCR4 signaling in the brain including disruption of the formation of new granule neurons in the adult brain.

DNA binding and dimerization determinants for thyroid hormone receptor alpha and its interaction with a nuclear protein.

The gel retardation assay was used to analyze the role of the thyroid hormone receptor alpha (TR alpha) ligand-binding domain (LBD) in controlling receptor interaction with a thyroid hormone responsive element (TRE). While wild type receptor TR alpha binds to the TRE mainly as monomer, deletion of 85 amino acids from its C-terminus results in a mutant receptor with enhanced DNA binding that forms several slow mobility complexes as revealed by gel retardation assay. Receptor deletion mutants that lack most of the LBD show significantly elevated DNA binding and are still able to bind to DNA as two complexes. Thus, the C-terminal end of TR alpha appears to interfere with the dimerization/oligomerization function and DNA binding of TR alpha. All C-terminal deletion mutants have lost their T3-responsive activator function, but some show constitutive activity. Nuclear factor from several cell lines, including CV-1, F9, and GC cells, interacts with TR alpha receptor to form a larger molecular weight complex as determined by gel retardation assay. This factor could not be detected in HeLatk- cells, where TR alpha does not activate a TRE-containing reporter gene. The nuclear factor is heat sensitive and does not bind to TRE itself but can interact with TR alpha in the absence of DNA. Deletion analysis demonstrates that the leucine zipper-like sequence located in the LBD of TR alpha is involved in this interaction. Together, our data suggest that TR alpha contains a dimerization function outside the LBD which is inhibited by the carboxy-terminal region, while the leucine zipper-like sequence in the LBD is required for interaction with a nuclear factor.

Surgical resection of jugulare foramen tumors by juxtacondylar approach without facial nerve transposition.

PRESENTATION: Jugulare foramen tumors (JFT) remain a difficult challenge especially in the forms extending extradurally and invading the petrous bone. In the standard technique, facial nerve function is placed at risks because of its transposition. We report on 31 extradural (N = 11) or intra-extra dural (N = 20) JFT resected surgically using the juxtacondylar approach alone or in combination with the infratemporal approach and without facial nerve transposition. RESULTS: The juxtacondylar approach permits the opening of the JF on its posteroinferior aspect and thus reduces the extent of petrous bone drilling. In tumors strictly located in the JF (N = 11), no petrous bone drilling is necessary and the facial nerve is never exposed. In tumors extending into the petrous bone (N = 20), the facial nerve was never transposed and moreover was kept in its bony canal in 15 cases. In only 5 cases, was the fallopian canal opened as the tumor was invading its bony wall. Similarly hearing function, when pre-operatively intact was always preserved and a better preservation of the lower cranial nerves could be achieved. Whatever the tumoral extent along the petrosal carotid artery, a radical resection could be realized in 30 cases. CONCLUSION: Therefore, we consider the juxtacondylar approach a useful adjunct to increase the possibilities of resection of JFT; it allows a better preservation of the neurovascular structures, especially the facial nerve which is kept in place and moreover kept in its bony canal when it is not invaded by the tumor.

Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors.

Thyroid hormones and retinoic acid function through nuclear receptors that belong to the steroid/thyroid-hormone receptor superfamily. Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) require auxiliary nuclear proteins for efficient DNA binding. Here we report that retinoid X receptors RXR alpha is one of these nuclear proteins. RXR alpha interacts both with TRs and with RARs, forming heterodimers in solution that strongly interact with a variety of T3/retinoic acid response elements. Transfection experiments show that RXR alpha can greatly enhance the transcriptional activity of TR and RAR at low retinoic acid concentrations that do not significantly activate RXR alpha itself. Thus, RXR alpha enhances the transcriptional activity of other receptors and its own ligand sensitivity by heterodimer formation. Our studies reveal a new subclass of receptors and a regulatory pathway controlling nuclear receptor activities by heterodimer formation.