safranine-t and Arthritis

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is known to inhibit matrix metalloproteinases, aggrecanases, and tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, ADAM17). These metalloproteases participate in different aspects of joint destruction in inflammatory arthritis. To determine the relative importance of this inhibitor in joint pathology, wild-type and Timp3-/- mice were immunized with methylated bovine serum albumin followed by arthritis induction by intra-articular injection of the same antigen. Animals were monitored for up to 14 days after challenge, and joint tissues were analyzed by routine and Safranin O staining and for the presence of aggrecan neoepitopes produced by metalloprotease cleavage. Serum TNF-alpha was measured by immunoassay. Compared to wild-type animals, Timp3-/- mice showed a dramatic increase in the initial inflammatory response to intra-articular antigen injection, and serum TNF-alpha levels were greatly elevated in the Timp3-/- animals after immunization. However, these differences in clinical features disappeared by days 7 to 14. No difference in Safranin O staining or aggrecan cleavage site neoepitope abundance was seen. Thus, in inflammatory joint disease TIMP-3 likely dampens the inflammatory response of TNF-alpha by reducing ADAM17 activity.

Topics: ADAM Proteins; ADAM17 Protein; Aggrecans; Animals; Antigens; Arthritis; Coloring Agents; Disease Models, Animal; Extracellular Matrix Proteins; Immunohistochemistry; Inflammation; Injections, Intra-Articular; Lectins, C-Type; Metalloendopeptidases; Mice; Mice, Knockout; Phenazines; Proteoglycans; Serum Albumin, Bovine; Tissue Inhibitor of Metalloproteinase-3; Tumor Necrosis Factor-alpha

To assess the clinical and histologic effects of an intraarticular application of low-dose (non-cytotoxic) liposomal clodronate in established antigen-induced monarthritis (AIA) in rabbits.. AIA was monitored by assessments of joint swelling, C-reactive protein levels, and radiographic changes in 17 NZW rabbits for 8 weeks during the course of weekly intraarticular injections of liposomal clodronate (0.145 mg/injection, low dose) or "empty" liposomes. The contralateral knee was injected with liposome buffer alone as the control. End-point analyses included macroscopic joint examination, immuno- and TUNEL staining, Safranin O staining/microspectrophotometry, and tumor necrosis factor alpha (TNFalpha) convertase enzyme (TACE) inhibition assay.. Liposomal clodronate-treated rabbits showed a reduction and delay in joint swelling during the first 3 injections. Expression of matrix-bound (solubilized) TNFalpha, lining cell hyperplasia, and levels of RAM-11+ macrophages were low in the synovium of the liposomal clodronate treatment group, but the proportion of apoptotic lining cells was not affected. The radiologic score was low at the end of weeks 2 and 4, but at 8 weeks, no difference, compared with controls, was found in pannus formation or in the extent of joint erosion; also, joint swelling was higher than before initiation of treatment. Injections of liposomal clodronate prevented cartilage proteoglycan loss, which was significant in the superficial zone only. TACE activity was not inhibited by clodronate.. Liposomal clodronate had temporary antiinflammatory and antierosive effects on established AIA in rabbits. Over the long-term, the loss of cartilage proteoglycans was halted. This observed treatment effect may be related to the inhibition of TNFalpha production and processing in the synovium.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Antigens; Apoptosis; Arthritis; Body Weight; C-Reactive Protein; Cartilage; Clodronic Acid; Injections, Intra-Articular; Liposomes; Metalloendopeptidases; Microspectrophotometry; Phenazines; Proteoglycans; Rabbits; Synovial Membrane

The intensity of safranin 'O' staining is directly proportional to the proteoglycan content in normal cartilage. Safranin 'O' has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin 'O' staining, in both normal and arthritic tissues. In cartilage where safranin 'O' staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin 'O' is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Arthritis; Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix; Humans; Immunohistochemistry; Keratan Sulfate; Middle Aged; Osteoarthritis; Phenazines; Proteoglycans

The appearance of age-related ulcerative changes in the mouse mandibular condyle were evaluated by light and electron microscopy examinations. Fibrillations appeared along the articular surface and in deeper tissue regions, as early as at six months of age. Such changes were characterized by a marked loss of the tissue's cellularity and by a marked reduction in matrix metachromasia and safranin-0 staining. These microscopical changes were accompanied by a reduced reactivity for both ruthenium red and colloidal iron binding, as noted ultrastructurally. At the same time, increasing numbers of erythrocytes appeared to be adhered to the surface irregularities and were also found in deeper regions within the articular lesions. Using morphological criteria, it became apparent that the degenerative changes of aging articular cartilage started at the more superficial regions of the tissue and only thereafter proceeded toward the chondro-osseous junction. Also, with the advancement of age, the degenerative changes became more severe.

Topics: Aging; Animals; Arthritis; Cartilage, Articular; Glycosaminoglycans; Mandibular Condyle; Mice; Mice, Inbred ICR; Phenazines; Rodent Diseases; Ruthenium Red; Tolonium Chloride

Arthritis was experimentally induced in the intercarpal joints of a series of mature ponies by the intraarticular injections of 400 microgram of the polyene antibiotic filipin in 1 ml of dimethyl sulfoxide. Twelve consecutive weekly injections were administered and the ponies were euthanatized 4 weeks after the last injection of filipin was made. The ponies were exercised for 1 hour each day throughout the experiment. Articular cartilage specimens from 4 sites in each intercarpal joint were examined histologically and histo-chemically. For the histochemical examination, safranin O-fast green, Alcian blue in 0.4 M and 0.9 M MgCl2 and Alcian blue 0.9 M MgCl2-Van Gieson matrical staining techniques were used. Decalcified sagittal sections of selected carpi were also examined histologically. There was superficial fibrillation with chondrocyte necrosis in the articular cartilage specimens of the joints. This was accompanied by loss of histochemical staining of the amorphous intercellular matrix, which was attributed to loss of glycosaminoglycans. There were hypertrophy of remaining chondrocytes and chondrone formation. On examination of decalcified sections of carpi, both exostoses and marginal osteophytosis was observed. There was evidence of both intramembraneous and endochondral osteogeneses in the exostoses. The pathologic changes seen in the experimental model were compared with those observed in the naturally occurring disease. It was concluded that the changes were comparable with the early changes of degenerative joint disease in the horse.

Topics: Alcian Blue; Animals; Arthritis; Carpus, Animal; Cartilage, Articular; Chlorides; Filipin; Forelimb; Hematoxylin; Horse Diseases; Horses; Magnesium; Phenazines