Jeffrey Kartchner and Dr. David Kooyman, Physiology and Developmental Biology
INTRODUCTION
As the population in the US continues to age, osteoarthritis (OA) is becoming a larger issue, currently effecting over 27 million people1. Presently, pharmaceutical options aim to lessen the effects of the disease, while no preventative treatment is currently available. Much of the current arthritis research is targeted at the effects of inflammation on the progression of OA. Our lab has shown in previous research that mice lacking the Receptor for Advanced Glycation End Products (RAGE) are protected from OA2. Other labs have shown the possibility of Toll-like receptor 4 (TLR-4) involvement in the progression of the disease as well3. The goal of this project was to determine the effects of blocking TLR-4 and the RAGE pathways in the progression of (OA). The data presented in this report are preliminary, due to the augmentation of the proposed experimental design to include additional controls. This expansion has set back our initial timetable.
MATERIALS/METHODS
Destabilization of the medial meniscus (DMM) surgeries were performed on RAGE KO mice and wild type mice matched in age and gender to induce OA. Based on observations from previous studies by our lab, surgeries were performed on mice that were 8-10 weeks old. CLI-095, a TLR-4 blocker, was suspended in DMSO and administered to both WT and RAGE KO mice knees at different concentrations (0.1 μM, 1.0 μM, 5.0 μM) topically at days 0, 7, 14, and 21 post-surgery. As a comparison control, DMSO without CLI-095 was applied in the same manner and time points to both RAGE KO and WT mice. At 28-days post-surgery, mice were sacrificed and their joint tissues harvested. This is a time point previously reported by us and others in which OA is clearly present after surgery2. We also have previously displayed that the biomarkers associated with OA do not present themselves until 14 days post-surgery. Tissues were decalcified, after which they were embedded in paraffin wax and sectioned for further analysis. Tissues were stained with Safranin O/Fast Green and scored using the OARSI scoring system, which determines the severity of the OA on a scale from 0-6, with 6 being the worst possible score for OA.
RESULTS
At this time the data collected is insufficient to form significant conclusions. Surprisingly, our initial analysis of these preliminary results shows that treating mice with CLI-095 has a deleterious effect on knee cartilage (Fig. 1). It does appear that our previous findings that RAGE KO mice are protected against OA have been reaffirmed, even when performing surgeries at a later time point (8-10 week old mice rather than 4-6 week old mice), as total OARSI scores for WT mice are higher than those of the RAGE KO mice (Fig. 2).
DISCUSSION
As the results obtained up to this point are insufficient to form conclusions, we plan to proceed with our experimental design to increase sample sizes.
REFERENCES
- R.C. Lawrence, D.T. Felson, C.G. Helmick, L.M. Arnold, H. Choi, R.A. Deyo, S. Gabriel, R. Hirsch, M.C. Hochberg, G.G. Hunder, J.M. Jordan, J.N. Katz, H.M. Kremers, F. Wolfe. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum, 58 (2008), pp. 26–35.
- J. Larkin, J.Z. Kartchner, D.M. Peterson, A.S. Doxey, W.R. Hollis, J.L. Rees, C. Ingersoll, G.G. Jackson, S.K. Wilhelm, S.S. Haynie, E. Chavez, P. Reynolds, D.L. Kooyman. The temporal expression of biomarkers associated with osteoarthritis after knee destabilizing surgery in mice with and without Receptor for Advanced Glycation End products (RAGE). Frontiers in Physiology, 4 (2013).
- P.L.E.M. van Lent, A.B. Blom, L. Grevers, A. Sloetjes, W.B. van den Berg. Toll-like receptor 4 induced FcgammaR expression potentiates early onset of joint inflammation and cartilage destruction during immune complex arthritis: Toll-like receptor 4 largely regulates FcgammaR expression by interleukin 10. Ann Rheum Dis, 66 (2007), pp. 334–340.