OBJECTIVE: To characterize the time course of aggrecan and type II collagen degradation in the rat iodoacetate model of cartilage degeneration in relationship to the temporal sequence that has been described in human osteoarthritis (OA). DESIGN: Rats were injected intra-articularly in one knee joint with iodoacetate and damage to the tibial plateau was assessed from digitized images captured using an image analyzer. The articular cartilage from the tibial plateau was harvested, extracted and glycosaminoglycan (GAG) content was measured using the dimethylmethylene blue (DMMB) assay. Cartilage aggrecan neoepitopes were detected in cartilage extracts by Western blotting using antibodies recognizing the aggrecanase-generated C-terminal neoepitope NITEGE (BC-13) and the MMP-generated C-terminal neoepitope DIPEN (BC-4). A type II collagen collagenase-generated neoepitope was detected in cartilage extracts by ELISA using the Col2-3/4Cshort antibody; denatured collagen was detected using the Col2-3/4m antibody. RESULTS: Degenerative joint changes and proteoglycan (GAG) loss progressed with time after iodoacetate injection. Western blotting of cartilage extracts of iodoacetate treated rats demonstrated an increase in both aggrecanase- and MMP-generated epitopes with the NITEGE aggrecanase neoepitope being significantly elevated on days 7, 14 and 21 while DIPEN the MMP neoepitope was significantly elevated on days 7 and 14. The type II collagen neoepitope recognized by Col2-3/4Cshort was significantly increased in cartilage extracts of rats at days 14 and 21 after iodoacetate injection. CONCLUSION: The proteoglycan fragments extracted from the knee cartilage of rats after the intra-articular injection of iodoacetate appeared to result from cleavage at both aggrecanase and MMP sites. Cleavage of type II collagen by collagenase was also detected after iodoacetate injection and occurred subsequent to the initiation of aggrecan loss. These observations serve to demonstrate similarities in the mechanisms of cartilage degeneration induced by iodoacetate to those seen in articular cartilage in OA.
OBJECTIVE: To characterize the time course of aggrecan and type II collagen degradation in the ratiodoacetate model of cartilage degeneration in relationship to the temporal sequence that has been described in humanosteoarthritis (OA). DESIGN:Rats were injected intra-articularly in one knee joint with iodoacetate and damage to the tibial plateau was assessed from digitized images captured using an image analyzer. The articular cartilage from the tibial plateau was harvested, extracted and glycosaminoglycan (GAG) content was measured using the dimethylmethylene blue (DMMB) assay. Cartilage aggrecan neoepitopes were detected in cartilage extracts by Western blotting using antibodies recognizing the aggrecanase-generated C-terminal neoepitope NITEGE (BC-13) and the MMP-generated C-terminal neoepitope DIPEN (BC-4). A type II collagen collagenase-generated neoepitope was detected in cartilage extracts by ELISA using the Col2-3/4Cshort antibody; denatured collagen was detected using the Col2-3/4m antibody. RESULTS: Degenerative joint changes and proteoglycan (GAG) loss progressed with time after iodoacetate injection. Western blotting of cartilage extracts of iodoacetate treated rats demonstrated an increase in both aggrecanase- and MMP-generated epitopes with the NITEGE aggrecanase neoepitope being significantly elevated on days 7, 14 and 21 while DIPEN the MMP neoepitope was significantly elevated on days 7 and 14. The type II collagen neoepitope recognized by Col2-3/4Cshort was significantly increased in cartilage extracts of rats at days 14 and 21 after iodoacetate injection. CONCLUSION: The proteoglycan fragments extracted from the knee cartilage of rats after the intra-articular injection of iodoacetate appeared to result from cleavage at both aggrecanase and MMP sites. Cleavage of type II collagen by collagenase was also detected after iodoacetate injection and occurred subsequent to the initiation of aggrecan loss. These observations serve to demonstrate similarities in the mechanisms of cartilage degeneration induced by iodoacetate to those seen in articular cartilage in OA.
Authors: Heather Stanton; Suzanne B Golub; Fraser M Rogerson; Karena Last; Christopher B Little; Amanda J Fosang Journal: Nat Protoc Date: 2011-03-03 Impact factor: 13.491
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