Immunohistochemical analysis of an equine model of synovitis-induced arthritis

P. G. Todhunter From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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S. A. Kincaid From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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R. J. Todhunter From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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J. R. Kammermann From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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B. Johnstone From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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A. N. Baird From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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R. R. Hanson From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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J. M. Wright From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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H.-C. Lin From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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R. C. Purohit From the Departments of Large Animal Surgery and Medicine (PG Todhunter, Baird, Hanson, Lin, Purohit), Anatomy and Histology (Kincaid, Kammermann), and Pathobiology (Wright), College of Veterinary Medicine, Auburn University, AL, 36849; Department of Clinical Sciences, Cornell University, Ithaca, NY, 14853 (RJ Todhunter); and Department of Biology, Case Western Reserve University, Cleveland, OH 44106 (Johnstone).

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Abstract

Objectives

To use lipopolysaccharide (LPS) to create synovitis in the midcarpal joint of ponies, and to assess the morphologic, histochemical, and immunohistochemical effects of synovitis on articular cartilage of the third carpal bone.

Animals

2- to 3-year-old ponies, 6 control (group 1) and 6 treated (group 2).

Procedure

Synovitis was induced in 1 midcarpal joint of group-2 ponies by intra-articular injections of LPS (0.02 μg/kg of body weight), morphine (0.1 mg/kg), and saline solution (group 2a) and morphine and saline solution alone in the contralateral midcarpal joint (group 2b). Articular cartilage sections and attached synovial membrane from the third carpal bones were examined by immunohistochemical distribution of interleukin 1β, tumor necrosis factor (TNF)-α, TNF receptors (P55, P75) and 3-B-3(–) epitopes, and by localization of proteoglycans (metachromatic staining). Proteoglycan extracts were assessed by metachromatic staining or western blotting and immunohistochemical staining, using anti-3-B-3 antibodies.

Results

Enhanced immunoreactivity for the cytokines and receptors was found in inflamed synovial membrane and noncalcified cartilage (group 2a more than 2b). Metachromasia of the noncalcified cartilage was greater in group-1 than in group-2a and group-2b specimens. In group 2a, chondrocyte hypertrophy and enhanced immunoreactivity for 3-B-3(–) epitope in areas of increased cytokine immunoreactivity suggested possible phenotypic change of the chondrocytes in response to synovitis. Immunohistochemical analysis by western blotting of proteoglycan extracts indicated strong 3-B-3(–) epitope immunolocalization in group-2a, weaker staining in group-2b, and barely detectable stain in group-1 specimens, which correlated with in situ immunolocalization.

Conclusions

Intra-articular administration of LPS may be used to induce a synovial environment conducive to increased immunoreactivity of interleukin 1β, TNF-α, and its receptors in equine synovial membrane and articular cartilage. These cytokines may be involved in the early phenotypic change of chondrocytes that is believed to occur in osteoarthritis and is characterized in this study by enhanced 3-B-3(–) epitope immunoreactivity and chondrocyte hypertrophy. (Am J Vet Res 1996;57:1080–1093)

Abstract

Objectives

To use lipopolysaccharide (LPS) to create synovitis in the midcarpal joint of ponies, and to assess the morphologic, histochemical, and immunohistochemical effects of synovitis on articular cartilage of the third carpal bone.

Animals

2- to 3-year-old ponies, 6 control (group 1) and 6 treated (group 2).

Procedure

Synovitis was induced in 1 midcarpal joint of group-2 ponies by intra-articular injections of LPS (0.02 μg/kg of body weight), morphine (0.1 mg/kg), and saline solution (group 2a) and morphine and saline solution alone in the contralateral midcarpal joint (group 2b). Articular cartilage sections and attached synovial membrane from the third carpal bones were examined by immunohistochemical distribution of interleukin 1β, tumor necrosis factor (TNF)-α, TNF receptors (P55, P75) and 3-B-3(–) epitopes, and by localization of proteoglycans (metachromatic staining). Proteoglycan extracts were assessed by metachromatic staining or western blotting and immunohistochemical staining, using anti-3-B-3 antibodies.

Results

Enhanced immunoreactivity for the cytokines and receptors was found in inflamed synovial membrane and noncalcified cartilage (group 2a more than 2b). Metachromasia of the noncalcified cartilage was greater in group-1 than in group-2a and group-2b specimens. In group 2a, chondrocyte hypertrophy and enhanced immunoreactivity for 3-B-3(–) epitope in areas of increased cytokine immunoreactivity suggested possible phenotypic change of the chondrocytes in response to synovitis. Immunohistochemical analysis by western blotting of proteoglycan extracts indicated strong 3-B-3(–) epitope immunolocalization in group-2a, weaker staining in group-2b, and barely detectable stain in group-1 specimens, which correlated with in situ immunolocalization.

Conclusions

Intra-articular administration of LPS may be used to induce a synovial environment conducive to increased immunoreactivity of interleukin 1β, TNF-α, and its receptors in equine synovial membrane and articular cartilage. These cytokines may be involved in the early phenotypic change of chondrocytes that is believed to occur in osteoarthritis and is characterized in this study by enhanced 3-B-3(–) epitope immunoreactivity and chondrocyte hypertrophy. (Am J Vet Res 1996;57:1080–1093)

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