Intra-articular injection of tumor necrosis factor-α in the rat: an acute and reversible in vivo model of cartilage proteoglycan degradation A.M. Malfait,

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Intra-articular injection of tumor necrosis factor-α in the rat: an acute and reversible in vivo model of cartilage proteoglycan degradation A.M. Malfait, M. Tortorella, J. Thompson, R. Hills, D.M. Meyer, B.D. Jaffee, K. Chinn, N. Ghoreishi-Haack, S. Markosyan, E.C. Arner Osteoarthritis and Cartilage Volume 17, Issue 5, Pages 627-635 (May 2009) DOI: 10.1016/j.joca.2008.10.005 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Time-course of cartilage aggrecan depletion. Rats were injected IA with 10μg TNFα. At various times following challenge, groups of rats were euthanized at the specified time-points and the knee joints were collected for Safranin O staining. “Time 0” represents uninjected controls. Maximal depletion of aggrecan occurred 16h after challenge, followed by partial replacement of proteoglycans, first in the tibial plateau (arrow, 24h) and later in the femoral condyles. Each rat knee shown is representative for a group of n=6. Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Assessment of aggrecanase-generated NITEGE neoepitope. Rats were injected IA with 10μg TNFα, and 16h later, knee joints were collected for histology compared with time 0 (uninjected controls). (A) Safranin O staining shows proteoglycan depletion in the femoral condyle; (B) immunohistochemistry reveals the presence of the NITEGE neoepitope in the depleted areas (blue signal, arrows). M=meniscus. Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Time-course of TNFα-induced release of GAG and aggrecanase-generated fragments into the SF. Rats were injected IA with 10μg TNFα [■] or with vehicle [●] and euthanized at various times (1, 2, 4, 6, 8, 16, 24 and 48h) following challenge. (A) GAG levels in the synovial lavage fluid were determined as μg GAG per ml of synovial lavage fluid (n=6 per group±SD); (B) Lavage fluids of n=6 rats were pooled by group and analyzed for the presence of the ARG neoepitope. Western blots of pooled samples at different time-points are shown (top) and the sum-density of the bands was measured by scanning densitometry (bottom). Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of indomethacin and dexamethasone on TNFα-induced proteoglycan degradation. Rats were injected IA with 10μg TNFα or with vehicle. Immediately prior to TNFα challenge, animals were treated orally with dexamethasone (1mg/kg), indomethacin (10mg/kg) or with vehicle. Sixteen hours following challenge, rats were euthanized and the joints were washed with 100μl saline. GAG levels in the synovial lavage fluid were measured, and results are presented as μg GAG per ml of synovial lavage fluid (n=6 per group±SD). Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Dose-related inhibition of TNFα-induced proteoglycan degradation by dexamethasone. Rats were injected IA with 10μg TNFα or vehicle. Immediately following TNFα challenge, animals were treated by continuous IV infusion with various doses of dexamethasone (0.001, 0.01, and 0.1mg/ml, translating to doses of 1.67, 16.7, and 167μg/kg/h, respectively) or with vehicle for the duration of the study and euthanized 16h following challenge. (A) GAG levels in the SF were determined on individual animals (n=6/group) as μg GAG per ml lavage fluid. The mean for each Dex-treated group (n=6) was then determined and % inhibition calculated compared to the mean GAG level in vehicle-treated TNFα positive control group. These % inhibition values were then plotted to determine the ED50 for inhibition by Dex; (B) Lavage fluids of n=6 rats were pooled and analyzed by Western blot for the presence of the ARG neoepitope; (C) ARG Western blots from several separate studies were quantified by scanning densitometry and percent inhibition of ARG epitope generation was plotted vs percent inhibition of GAG release for each group. Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Assessment of aggrecanase-generated SELE neoepitope in synovial lavage fluids. Rats were injected IA with 10μg TNFα, euthanized 6h following challenge and lavage samples pooled. (A) Various volumes of lavage fluid were assessed for SELE and ARG-containing neoepitopes; (B) Replicate lanes were run by SDS-PAGE and then blots cut and assessed by SELE, ARG and FFGV neoepitope Western analysis. Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Time-course of TNFα-induced release of aggrecan neoepitopes, SELE and AGEG. Rats were injected IA with 10μg TNFα and groups of rats were euthanized at various times following challenge. (A). Lavage fluids of n=6 rats were pooled and analyzed for the presence of the SELE and AGEG neoepitopes by Western blot. (B) GAG levels in of the SF lavages were measured and AGEG and SELE blots were quantified by scanning densitometry. Data are plotted as percent of maximal level vs time following TNFα challenge. Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Inhibition of TNFα-induced aggrecan degradation with an aggrecanase inhibitor. Rats were injected IA with vehicle or 10μg of TNFα, with or without continuous IV infusion of an aggrecanase inhibitor. At 6h following challenge, rats were euthanized, synovial lavage fluids collected and knee joints taken for histology. (A) Knee joints were stained with Safranin O. Shown is the median representative of n=5 for control, n=7 for TNFα, and n=6 for TNF+drug groups; (B) synovial lavage fluids of individual rats were analyzed for the presence of aggrecan fragments with the new N-terminus AGEG. Western blots (shown on top) were quantified by scanning densitometry and data are presented as mean±s.e.m. of antibody reactivity. Osteoarthritis and Cartilage 2009 17, 627-635DOI: (10.1016/j.joca.2008.10.005) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions