Objective To investigate the relationship between NF-κB activity, cytokine levels, and pain sensitivities in a rodent model of osteoarthritis (OA). Method OA was induced in transgenic NF-κB luciferase reporter mice via mono-iodoacetate (MIA) intra-articular injection. Using luminescent imaging we evaluated the temporal kinetics of NF-κB activity and its relationship to the development of pain sensitivities and serum cytokine levels in this model. Results MIA induced a transient increase in joint-related NF-κB activity at early time points (day 3 post-injection) and an associated biphasic pain (mechanical allodynia) response. NF-κB activity, serum IL-6, IL-1β, and IL-10 accounted for ~75% of the variability in pain-related mechanical sensitivities in this model. Specifically, NF-κB activity was strongly correlated to mechanical allodynia and serum IL-6 levels in the inflammatory pain phase of this model (day 3), while serum IL-1β was strongly correlated to pain sensitivities in the chronic pain phase of the model (day 28). Conclusion Our findings suggest that NF-κB activity, IL-6 and IL-1β may be playing distinct roles in pain sensitivity development in this model of arthritis and may act to distinguish the acute from chronic pain phases of this model. This work establishes luminescent imaging of NF-κB activity as a novel imaging biomarker of pain sensitivities in this model of OA.

Large macromolecules were retained in the arthritic joint longer than in the healthy joint, while smaller molecules were cleared similarly in healthy and arthritic joints. In vivo fluorescence imaging, plasma and lymph node concentrations, and spatial distributions of drug fluorescence identified differences in higher molecular weight clearance between naive and arthritic disease states. Findings may relate to a thickening of synovium for joints with induced arthritis, and support the concept that intra-articular drug delivery effectiveness may vary with the state of joint pathology.

Objective To determine the utility of silk fibroin (SF) microparticles as sustained release vehicles for intra-articular delivery. Design SF formulations were varied to generate microparticle drug carriers that were characterized in vitro for their physical properties, release kinetics for a conjugated fluorophore (Cy7), and in vivo for intra-articular retention time using live-animal, fluorescence in vivo imaging. Results SF microparticle carriers were spherical in shape and ranged from 598 nm to 21.5 μm in diameter. SF microparticles provided for sustained release of Cy7 in vitro, with only 10% of the initial load released over 7 days. Upon intra-articular injection in rat knee joints, the SF microparticles were associated with an intra-articular fluorescence decay half-life of 43.3 hours, greatly increasing the joint residence over that for an equivalent concentration of SF-Cy7 in solution form. The SF microparticles also increase the localization of dye within the joint cavity as determined by image analysis of fluorescent gradients, significantly reducing distribution of the Cy7 to neighboring tissue as compared to SF-Cy7 in free solution. Conclusion Silk microparticles act to provide for localized and sustained delivery of loaded small molecules following intra-articular injection, and may be an attractive strategy for delivering small molecule drugs for the treatment of arthritis.

Objective: Osteoarthritis (OA) is a consequence of not only mechanical events such as joint instability, but also biological events that result in the upregulation of proinflammatory and catabolic mediators. The intra-articular injection of monoiodoacetate (MIA) has been widely used to induce OA. NF-B activity has been linked to increased expression of proinflammatory cytokines (IL-1, TNF-, IL-6, etc), metalloproteinases (MMPs), chemokines and inducible enzymes, which all contribute to cartilage degradation and subsequent OA. The goal of this study was to use in vivo imaging (IVIS) of NF-B activation to track longitudinal changes due to inflammation in a rodent model of OA. Design: Twenty-four (24) NF-B-luc reporter transgenic mice [BALB/C-Tg (NF-B-RE-luc)-Xen, age 7-8 weeks] were given intra-articular knee injections with either MIA (n = 12) or normal saline (n = 12) to serve as a control. IVIS and ex vivo imaging of NF-B and tactile allodynia measurements were performed, and correlations were recorded preoperatively and on days 1, 3, 7, 14, 21 and 28. Animals were euthanized on days 3 and 28 for ex vivo imaging, and tissues were stored for future immunohistochemical evaluation. Results: NF-B activity was significantly elevated in the MIA group on days 1 and 3 (p < 0.05) when compared to preoperative levels and was significantly elevated compared to the normal saline group on day 3 (p < 0.05). There was a significant increase in tactile allodynia in the MIA group compared to preoperative levels, as well as compared to the normal saline group at all time points (p < 0.05). In vivo NF-B luminescence correlated with tactile allodynia (p < 0.0001) and with ex vivo imaging (p < 0.0001). Conclusion: This study validates the use of IVIS imaging of NF-B activity in a MIA rodent model of arthritis and provides evidence for the use of NF-B luminescence imaging as an imaging biomarker of pain sensitivities. This can be utilized in the future to further elucidate NF-B's role in inflammation and OA. In addition, it can help evaluate potential therapeutic agents that target NF-B.

locomotion. Ccr2 null mice were protected from these locomotion decreases, despite similar joint damage at 8 weeks post DMM, and Ccr2 null DRG did not produce MCP-1. Therefore, we sought to further investigate the expression and regulation of MCP-1 in the DMM model. The current goals were to: 1) Test whether TNF-a induces MCP-1 production in DRG cells, since it has been shown that TNFa induces Mcp-1 mRNA in cultured DRG cell lines. 2) Analyze MCP-1 protein content in the knees of wild-type (WT) and Ccr2 null mice after DMM. Methods: Knee-innervating DRG, L3-L5, were collected from 10-week old naïve C57BL/6 WT or Ccr2 null mice. Cells were isolated and cultured for 2 days in basal medium before 48 h-stimulation with 0, 25, or 100 ng/mL TNF-a; supernatants were collected for MCP-1 ELISA. Hip cartilage explants were harvested from 5-week old naove C57BL/6 mice. Hip cartilage was used since it is not possible to culture mouse knee cartilage explants. Explants were rested overnight, stimulated with 100 ng/mL TNF-a for 48 h, and supernatants were collected for MCP-1 ELISA. DMM or sham surgery was performed in the right knees of 10-week old male C57BL/6 WT or Ccr2 null (Taconic #3736) mice. At 0, 4, and 8 weeks after surgery, whole knee joints were extracted for ELISA. At 4 or 8 weeks post surgery, DRG cells were cultured for 4 days and supernatants collected for ELISA. Results: In order to test whether TNF-a is able to stimulate primary DRG cells to produce MCP-1 protein, we treated naïve WT DRG cells with TNF-a. Cultures stimulated with 25 ng/mL TNF-a produced 37-fold increased amounts of MCP-1 compared to unstimulated cells (p<0.0001); a higher concentration of 100 ng/mL TNF-a did not further increase MCP-1. This confirms earlier reports of increased Mcp-1 mRNA following TNF-a stimulation. DRG cells from naïve Ccr2 null mice responded in the same way to TNF-a. We looked for the presence of TNF-a in cultures of DRG cells taken from WT or Ccr2 null naïve, sham, or DMM mice at 4 and 8 weeks post surgery. None of these cultures contained measurable amounts of TNF-a. Next, we determined MCP-1 levels in whole knee joint extracts after DMM in WT and Ccr2 null mice. In WT, knee MCP-1 protein levels were elevated 4 weeks post DMM, compared to sham and naïve age-matched controls (p<0.0001); by 8 weeks post DMM, levels had returned to baseline. MCP-1 was also increased in knee extracts from Ccr2 null mice, 4 weeks after DMM, but to a lesser extent than in WT. In order to determine the potential source of MCP-1 in the knee, we began by testing whether cartilage is able to produce MCP-1. Cartilage explants stimulated with 100 ng/mL TNF-a produced 86-fold increased levels of MCP-1 compared to unstimulated explants (p¼0.0008). When we looked for the presence of TNF-a in knee extracts, however, we found that DMM and naïve extracts contained similar low levels of TNF-a at 4 and 8 weeks post surgery. TNF-a levels in Ccr2 null knee extracts mirrored the WT results, with no difference at 4 or 8 weeks between DMM and naïve extracts. Conc...