Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture

OBJECTIVE: To measure concentrations of nitric oxide metabolites (nitrite-nitrate [NOt]) in cartilage, synovial membrane, and cranial cruciate ligament (CCL) in dogs and evaluate associations with osteoarthritis in dogs with CCL rupture. ANIMALS: 46 dogs with CCL rupture and 54 control dogs without joint disease. PROCEDURE: Tissue specimens for histologic examination and explant culture were harvested during surgery in the CCL group or immediately after euthanasia in the control group; NOt concentrations were measured in supernatant of explant cultures and compared among dogs with various degrees of osteoarthritis and between dogs with and without CCL rupture. RESULTS: Osteoarthritic cartilage had significantly higher NOt concentration (1,171.6 nmol/g) than did healthy cartilage (491.0 nmol/g); NOt concentration was associated with severity of macroscopic and microscopic lesions. Synovial membrane NOt concentration did not differ between dogs with and without CCL rupture. Ruptured CCL produced less NOt than did intact ligaments. In control dogs, NOt concentrations were similar for intact ligaments (568.1 nmol/g) and articular cartilage (491.0 nmol/g). Synthesis of NOt was inhibited substantially by coincubation with inhibitors. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that NOt in canine joint tissues originates from the inducible nitric oxide synthase pathway. Nitric oxide metabolite production in cartilage was greater in dogs with osteoarthritis than in healthy dogs and was associated with lesion severity, suggesting that nitric oxide inhibitors may be considered as a treatment for osteoarthritis. The CCL produces substantial concentrations of NOt; the importance of this finding is unknown.     

Endogenous nitric oxide production in canine osteoarthritis: Detection in urine, serum, and synovial fluid specimens

OBJECTIVE: To measure nitric oxide (NO) concentrations in serum, urine, and synovial fluid (SF) of dogs with naturally occurring cranial cruciate ligament (CCL) rupture and normal dogs, and to compare these with clinical and histologic changes of osteoarthritis (OA). STUDY DESIGN: Prospective clinical study including 2 groups of animals selected from the hospital population. ANIMALS: Forty-three dogs (CCL group) with OA secondary to CCL rupture; 30 healthy dogs (control group) without CCL rupture. METHODS: Serum, urine, and SF were collected before and during surgery in the CCL group or immediately after euthanasia in the control group. Articular cartilage and synovial membrane tissue specimens were prepared for routine histologic examination. The stable end products of NO, total nitrite and nitrate (NOt) activity, were measured in body fluids and compared with macroscopic and histologic degrees of OA. Urinary NOt concentration was compared with urinary creatinine concentration and stated as urinary NOt:creatinine ratio (UNCR). RESULTS-SF NOt concentrations were not significantly different between the 2 groups. Serum NOt concentrations (45.6 vs 28.9 micromol/L; P =.042) and the UNCR (0.007 vs 0.004; P =.035) were significantly higher in dogs of the CCL group compared with the control population. An association between UNCR and histologic and macroscopical OA grades could be demonstrated. CONCLUSION: UNCR might be a useful indicator of nitrite and nitrate production and, therefore, osteoarthritic changes in joints. CLINICAL RELEVANCE: UNCR could be used as a tool to evaluate the NOt production by joint tissues over time and might therefore provide a method of evaluating the effects of drugs in the control of osteoarthritis.     

Detection and distribution of apoptotic cell death in normal and diseased canine cranial cruciate ligaments

One of the possible initiating factors in canine cranial cruciate ligament (CCL) rupture could be an abnormal pattern of ligament cell death. This study compared apoptotic cell death in sections of ruptured CCLs and normal controls, and examined nitric oxide (NO) production in joint tissues and correlated this to apoptosis. CCLs and cartilage from the lateral femoral condyle were harvested from 10 healthy dogs and 15 dogs with CCL rupture and ligaments were further processed to detect cleaved caspase-3 and to determine supernatant NO production in explant cultures. Apoptotic activity was greater in ruptured ligaments compared to controls. NO in ligaments showed a moderate but significant positive correlation with caspase-positive cells. The results suggest that increased apoptosis has a role in CCL rupture and that apoptosis may be influenced by local NO production.     

Prevalence and relevance of antibodies to type-I and -II collagen in synovial fluid of dogs with cranial cruciate ligament damage

OBJECTIVE: To measure and compare synovial fluid antibody titers to type-I and -II collagen in stifle joints with instability caused by complete or partial cranial cruciate ligament (CCL) rupture and joints with osteoarthrosis secondary to other pathologic changes in dogs. ANIMALS: 82 dogs with diseased stifle joints. PROCEDURE: Synovial fluid samples were collected from 7 dogs with clinically normal stifles (control group) and 82 dogs with diseased joints (50 stifle joints with complete rupture of the CCL, 20 with partial damage of the CCL, and 12 joints with radiographic signs of osteoarthritis secondary to other arthropathies). Synovial fluid samples were tested for autoantibodies to type-I and -II collagen by an ELISA. RESULTS: In dogs with complete and partial CCL rupture, synovial fluid antibody titers to type-I and -II collagen were significantly increased, compared with control dogs. Forty-eight percent (24/50) of samples from dogs with complete CCL rupture and 35% (7/20) of samples from dogs with partial CCL rupture had antibody titers to type-I collagen that were greater than the mean plus 2 standard deviations of the control group titers. Synovial fluid antibody titers to type-II collagen were high in 40% of the dogs with partial or (8/20) complete (20/50) CCL rupture. Dogs with osteoarthrosis secondary to other pathologic changes had significantly increased synovial fluid antibodies to type-I and -II collagen, compared with control dogs. CONCLUSION: Increases in autoantibodies to collagen in synovial fluid are not specific for the type of joint disorder. It is unlikely that the anticollagen antibodies play an active role in the initiation of weakening of the CCL.     

Cartilage-derived biomarkers of osteoarthritis in synovial fluid of dogs with naturally acquired rupture of the cranial cruciate ligament

OBJECTIVE: To compare synovial fluid biomarkers of cartilage metabolism in joints with naturally acquired or experimentally induced cranial cruciate ligament (CCL) rupture and determine correlations with stage and severity of disease in dogs. ANIMALS: 95 dogs with ruptured CCL, 8 dogs with experimentally ruptured CCL, and 24 healthy dogs. PROCEDURES: Synovial fluid was assayed for chondroitin sulfate neo-epitopes 3B3(-) and 7D4 and glycosaminoglycan (GAG) concentration. Results were correlated with demographic data, duration of lameness, radiographic osteoarthritis score, and intra-articular lesions. RESULTS: The 7D4 concentrations and 7D4:GAG in synovial fluid from joints with naturally acquired CCL rupture and experimental CCL transection were similar and significantly greater than values for healthy control joints. The 3B3(-) concentrations in the CCL-deficient groups were not significantly different, although only values in the naturally acquired CCL rupture group were significantly greater than those in the healthy control group. Within the naturally acquired CCL rupture group there was a significant correlation between 3B3(-) and 7D4 concentrations. However, there were no significant correlations between biomarker concentrations and continuous demographic or disease-related variables or differences in biomarker concentrations with different categories of disease. CONCLUSIONS AND CLINICAL RELEVANCE: Synovial fluid biomarker concentrations were significantly increased in joints with secondary osteoarthritis associated with naturally acquired or experimental CCL rupture; however, lack of apparently simple relationships with demographic variables or stage or severity of disease limits their clinical usefulness.     

Nitric oxide and metalloproteinases in canine articular ligaments: a comparison between the cranial cruciate, the medial genual collateral and the femoral head ligament

Osteoarthritis due to cranial cruciate ligament (CCL) rupture or hip dysplasia is one of the most important causes of chronic lameness in dogs. This study aimed at comparing nitric oxide (NO) production by the CCL with that of the femoral head ligament (FHL) and the medial collateral ligament (MCL), and investigating the pathway of NO production and the concomitant metalloproteinase (MMP) activity in the presence or absence of an inflammatory stimulus. Ligaments of normal dogs were subjected to different stimuli, and NO and MMP activity from explant culture supernatants were compared. The results showed that in explant cultures of the canine CCL more NO was produced than in those of the other two ligaments. A higher level of NO was produced when CCLs were exposed to the inducible nitric oxide synthase (iNOS)-inducing cocktail TNF/IL-1/LPS, and NO synthesis could be inhibited by both l-NMMA, a general nitric oxide synthase (NOS) inhibitor and l-NIL, a specific iNOS inhibitor. However, a correlation between NO synthesis and iNOS expression levels as determined by immunohistochemistry was not observed. In contrast to CCL, no evidence for iNOS-dependent NO synthesis was observed for MCL and FHL. The CCL produced less MMP than MCL and FHL, and no correlation between MMP and NO could be demonstrated. MMP activity in the CCL increased significantly after 48 h of incubation with the inflammatory stimulus. The results suggest that in canine osteoarthritis NO synthesized by canine CCL plays a more important role in the pathogenesis of osteoarthritis of the stifle than that synthesized by FHL and MCL.     

Evaluation of a Collagenase Generated Osteoarthritis Biomarker in Naturally Occurring Canine Cruciate Disease

Objective— To determine the clinical value of a novel osteoarthritis (OA) biomarker in detecting canine cruciate disease.
Study Design— Cross sectional clinical study.
Animals— Dogs (n=22) with cranial cruciate ligament (CCL) rupture and 12 control dogs.
Methods— Concentrations of collagenase-generated cleavage epitope of type II collagen (Col2-3/4C_{long mono}, or C2C) in serum, urine, and joint fluid were compared between a group of dogs with CCL rupture and a control group. Correlation of C2C concentrations to the clinical stage of stifle OA was also evaluated.
Results— There were no significant differences in C2C concentrations in serum, urine, and joint fluid between groups ( P >.05). Subjective scores of lameness, joint effusion, osteophytosis were significantly more severe in the CCL rupture group compared with the control group ( P <.05). There was no significant correlation of C2C concentrations with clinical stage of stifle OA ( P >.05).
Conclusion— This OA biomarker did not detect pathology associated with CCL rupture. Our results suggest that collagenase-specific degradation of type II collagen in articular cartilage may not be involved in the early stage of naturally occurring canine cruciate disease, and that pathology associated with naturally occurring CCL rupture is different from that of experimental OA model.
Clinical Relevance— C2C is not clinically useful in detecting CCL rupture in dogs.     

Pathologic changes in grossly normal menisci in dogs with rupture of the cranial cruciate ligament

OBJECTIVE: To determine whether histopathologic changes are detectable in grossly normal medial menisci from dogs with rupture of the cranial cruciate ligament (CCL). DESIGN: Case series. SAMPLE POPULATION: 40 medial menisci from dogs with rupture of the CCL and 20 medial menisci from control dogs without stifle joint disease. PROCEDURE: Data evaluated included age, duration of clinical signs, and whether rupture of the CCL was complete or incomplete. Three groups (n = 20/group) were also compared on the basis of 5 histologic criteria; group-1 menisci appeared grossly normal and were obtained from dogs with naturally occurring rupture of the CCL, group-2 menisci were grossly abnormal and were also obtained from dogs with naturally occurring CCL ruptures, and group-3 menisci were collected at postmortem from dogs without stifle joint disease that were of similar age and weight as dogs in groups 1 and 2. RESULTS: Group-2 menisci were significantly different from group-1 and -3 menisci in all histologic criteria. Group-1 menisci were significantly different from control menisci in only 1 of the 5 histologic criteria (cartilage differentiation). Dogs that were > or =3 years old had significantly more surface cellularity than did dogs that were < 3 years old. A significant difference was not detected between groups 1 and 2 with regard to completeness of rupture. CONCLUSIONS AND CLINICAL RELEVANCE: Histologic changes in meniscal cartilage correlate with gross appearance of the cartilage at time of surgery for rupture of the CCL. On the basis of minimal histologic changes, routine removal of grossly normal menisci does not appear to be warranted.     

Orally administered doxycycline accumulates in synovial fluid compared to plasma

Reasons for performing study: Tetracycline compounds have been used to slow the progression of osteoarthritis (OA) and rheumatoid arthritis but the concentration of doxycycline attained in synovial fluid following oral, low‐dose administration has yet to be determined. Objective: To determine the concentration of doxycycline in synovial fluid following oral, low‐dose administration. Methods: Six mature horses received doxycycline (5 mg/kg bwt q. 12 h for 5 doses). Venous blood and synovial fluid samples were collected at t = 0, 0.25, 0.5, 1, 12, 24, 48 and 72 h. Doxycycline concentrations were measured using reverse phase high pressure liquid chromatography with ultraviolet detection. Results: Doxycycline concentrations at all time points after t = 0 were above the lower limit of quantification for the assay. Plasma concentrations of doxycycline were above 0.21 µg/ml at t = 0.5 h. The mean ± s.d. peak concentration (C_max) of doxycycline in plasma was 0.37 ± 0.22 µg/ml and time to peak concentration was 0.54 ± 0.19 h. Synovial fluid concentrations of doxycycline were above 0.12 µg/ml 1 h after drug administration. The mean C_max of doxycycline in the synovial fluid was 0.27 ± 0.10 µg/ml. The penetration factor of doxycycline from plasma into synovial fluid, as determined by a ratio of the area‐under‐the‐curve for synovial fluid:plasma during the sampling period, was 4.6. Potential relevance: Orally administered doxycycline distributes easily into synovial fluid with a penetration factor of 4.6. Terminal half‐life of the drug in synovial fluid was longer than in the plasma, indicating possible accumulation in this compartment. Further in vivo studies are warranted to define a medication protocol prior to routine clinical use of doxycycline for the treatment of OA.     

Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints

Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance. Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity. Slight discolouration and fibrillation or minor clefts of the cartilage were considered as moderate OA, whereas erosions of articular cartilage down to the subchondral bone were considered as cases of severe OA. Explant cultures of equine synovial membrane and articular cartilage released the local mediators, NO and PGE2, as well as detectable levels of NMP activity into culture media. Concentrations of NO were higher in articular cartilage explants compared to synovial membrane explants, whereas concentrations of PGE2 were higher in synovial membrane explants. The NMPs with collagenolytic activities were similar in both explant cultures, whereas gelatinolytic activities were higher in synovial membrane explant cultures and caseinolytic activities were generally higher in articular cartilage explant cultures. Furthermore it was shown that concentrations or enzyme activities increased according to the severity of disease of the joints. Concentrations for NO, collagenolytic and gelatinolytic NMPs were relatively stable, whereas PGE2 and caseinolytic NMP concentrations increased over time in culture.     

Changes in the local regulation of insulin-like growth factors I and II and insulin-like growth factor-binding proteins in osteoarthritis of the canine stifle joint secondary to cruciate ligament rupture

OBJECTIVES: To investigate changes in concentrations of insulin-like growth factors I (IGF-I) and II (IGF-II) and the expression of IGF-binding proteins (IGFBP) in synovial fluids from dogs with naturally occurring osteoarthritis (OA) of the canine stifle joint secondary to cranial cruciate ligament (CCL) rupture. STUDY DESIGN: Prospective study with synovial fluid sampling from diseased and contralateral unaffected joints at 0, 1.5, and 5 months. SAMPLE POPULATION: Eleven dogs with unilateral CCL deficiency, with unaffected contralateral joints. METHODS: IGF-I and IGF-II concentrations in synovial fluids were estimated by radioimmunoassay at 0, 1.5, and 5 months; Western ligand blotting was performed for intact IGFBPs at 0, 1.5, 5, and 9 months. Both stifle joints were radiographed at 0, 7, and 13 months. RESULTS: The IGF system is altered after CCL rupture and during development of early OA. Mean IGF-I and IGF-II concentrations in index stifle joints at study entry were 201.6 microg/mL and 345.7 microg/mL, respectively, compared with 57.7 microg/mL and 79.4 microg/mL, respectively, for contralateral joints. Index joint IGF concentrations increased after surgical treatment and then declined, although they remained higher than contralateral joints. Index joints had increases in IGFBP-3 and -4, and a decrease in IGFBP-2 expression compared with contralateral joints. CONCLUSIONS: Although IGF concentrations are increased in canine OA, alterations in IGFBP profiles may limit the tissue availability of IGF. CLINICAL RELEVANCE: Manipulation of the IGF system may provide an opportunity for novel treatments of OA in dogs.     

Matrix metalloproteinase activity in stifle synovial fluid of cranial cruciate ligament deficient dogs and effect of postoperative doxycycline treatment

This prospective clinical study investigated the activity of matrix metalloproteinases (MMPs) in stifle synovial fluid (SF) of 13 dogs with acute cranial cruciate ligament (CCL) rupture, and the effect of a postoperative doxycycline treatment. MMP-2, 3, 9 and 13 activities were compared with respect to the time of sampling (preoperatively or 1 month after surgical stabilisation) and the type of postoperative adjuvant treatment (doxycycline or not). No significant activity was detected for both MMP-3 and MMP-13. MMP-2 and MMP-9 activities were found to be significantly highly increased in SF of CCL ruptured stifles compared to control stifles of unaffected dogs. No significant effect from surgical stabilisation and postoperative doxycycline treatment on MMP-2 and MMP-9 activities was found, indicating that doxycycline may not be an appropriate postoperative medical treatment after CCL rupture.     

Collagen fragmentation in ruptured canine cranial cruciate ligament explants

Collagen fragmentation in cranial cruciate ligament (CCL) explants and stifle synovial fluid was investigated in dogs with ruptured and intact CCL. Cathepsin K and tartrate-resistant acid phosphatase (TRAP) activities were determined in CCL explant supernatants. Formation of collagen fragments was determined in explant supernatants and stifle synovial fluid. Cathepsin K(+) and TRAP(+) cells were stained specifically in histological sections of CCL. Formation of telopeptide collagen fragments was increased in ruptured CCL explants and stifle synovial fluid from dogs with ruptured CCL. In ruptured CCL explants, release of collagen fragments was associated with extracellular release of TRAP and the presence of cathepsin K(+) cells within CCL tissue. Cathepsin K(+) and TRAP(+) cells were only seen in ruptured CCL. It was concluded that infiltration of the CCL with TRAP(+) cells in dogs with CCL rupture is associated with increased collagenolysis. It is hypothesized that recruitment and activation of TRAP(+) mononuclear cells within the synovium and CCL precipitates CCL rupture through upregulation of collagenolytic enzymes and collagen degradation.     

Immunohistochemical characterization of inflammatory cell populations and adhesion molecule expression in synovial membranes from dogs with spontaneous cranial cruciate ligament rupture

This study describes the distribution of CD4+ and CD8alpha+ T lymphocytes, B lymphocytes, macrophages, MHC class II antigens, immunoglobulin (IgG, IgM, IgA)-containing cells and of adhesion molecules belonging to the CD11/CD18 family in synovial membrane biopsies from 28 dogs with spontaneous rupture of the cranial cruciate ligament (CCL). Synovial membranes from 11 dogs without evidence of joint lesions were used as control tissues. The main cell types in synovial membranes from dogs with CCL rupture were B lymphocytes and plasma cells belonging to the IgG isotype. The severity of inflammatory cell infiltration in CCL cases was positively correlated with the expression of adhesion molecules. Double immunofluorescence labelling of frozen sections revealed that in the inflamed synovium of dogs with CCL rupture numerous dendritic cells expressing MHC class II antigen and canine CD1c were present. The findings further support the view that in the synovium of dogs with CCL rupture an immunologic response is going on in which dendritic cells are possibly involved by presenting hitherto unknown antigens to T lymphocytes.     

Concentrations of chondroitin sulfate epitopes 3B3 and 7D4 in synovial fluid after intra-articular and extracapsular reconstruction of the cranial cruciate ligament in dogs

OBJECTIVE: To evaluate effects of intra-articular and extracapsular reconstruction of the cranial cruciate ligament (CCL) on metabolism of articular cartilage as reflected by concentrations of chondroitin sulfate epitopes 3B3 and 7D4 in synovial fluid. ANIMALS: 13 adult dogs. PROCEDURE: Each dog underwent unilateral CCL transection (CCLT). One month after CCLT, sham CCL reconstruction (3 dogs), intra-articular CCL reconstruction (5), or extracapsular CCL reconstruction (5) was performed. Synovial fluid was collected by direct arthrocentesis from CCLT and contralateral stifle joints immediately before (time 0) and 1, 3, and 5 months after CCLT. Fluid was examined for concentrations of 3B3 and 7D4 epitopes and total sulfated glycosaminoglycan (GAG) content. RESULTS: Concentrations of 3B3, 7D4, and GAG, 3B3:GAG, or 7D4:GAG in CCLT joints did not differ significantly among treatment groups nor in the ratios of these variables in CCLT joints to contralateral joints at 3 months. In a longitudinal analysis, concentrations of 3B3 and 7D4, 3B3:GAG, and 7D4:GAG in CCLT joints in all groups changed significantly with time, but we did not detect time X group interactions. CONCLUSIONS AND CLINICAL RELEVANCE: Transection of CCL resulted in significant perturbation in articular cartilage metabolism as reflected by alterations in concentrations of 3B3 and 7D4 in synovial fluid. These changes over time were not significantly influenced by method of CCL reconstruction. We did not find evidence that surgical stabilization of CCL-deficient joints by intra-articular or extracapsular techniques had any effect on preventing alterations in composition of synovial fluid that have been associated with secondary osteoarthritis.     

The contribution of the synovium, synovial derived inflammatory cytokines and neuropeptides to the pathogenesis of osteoarthritis

Osteoarthritis (OA) is one of the most common and disabling chronic joint disorders affecting horses, dogs and humans. Synovial inflammation or synovitis is a frequently observed phenomenon in osteoarthritic joints and contributes to the pathogenesis of OA through formation of various catabolic and pro-inflammatory mediators altering the balance of cartilage matrix degradation and repair. Catabolic mediators produced by the inflamed synovium include pro-inflammatory cytokines, nitric oxide, prostaglandin E(2) and several neuropeptides, which further contribute to the pathogenesis of OA by increasing cartilage degradation. Recent studies suggest that substance P, corticotropin-releasing factor, urocortin and vasoactive intestinal peptide may also be involved in OA development, but the precise role of these neuropeptides in the pathogenesis of OA is not known. Since increased production of matrix metalloproteinases by the synovium is stimulated by pro-inflammatory cytokines, future anti-inflammatory therapies should focus on the synovium as a means of controlling subsequent inflammatory damage.     

Comparison of tibial plateau angles in normal and cranial cruciate deficient stifles of Labrador retrievers

OBJECTIVE: To investigate tibial plateau angles (TPA) in normal and cranial cruciate ligament (CCL) deficient stifles of Labrador retrievers. STUDY DESIGN: Prospective clinical study. ANIMALS: Eighty-one client-owned purebred Labrador retrievers. METHODS: Lateral radiographs of the tibia were obtained from 2 groups of dogs. Group I (42 dogs) had CCL rupture diagnosed by arthrotomy or arthroscopy. Group II (39 dogs) had no history of orthopedic problems, no radiographic evidence of CCL rupture, and dogs were >8 years of age. The tibial axis and the tibial plateau were determined on the radiographs, and the TPA was measured using image measurement software. The TPA measurement results of groups I and II were compared. RESULTS: Group I (CCL rupture) had a mean TPA (+/-SD) of 23.5 (+/-3.1) degrees, and group II (normal) had a mean TPA (+/-SD) of 23.6 (+/-3.5) degrees. With a P value of.97, no statistical difference was detected between the 2 groups. CONCLUSIONS: No correlation between the magnitude of TPA and CCL rupture was identified in this group of Labrador retrievers. CLINICAL RELEVANCE: In Labrador retrievers, TPA should not be used as a predictor of CCL rupture.     

Immunocytochemical demonstration of lymphocyte subsets and MHC class II antigen expression in synovial membranes from dogs with rheumatoid arthritis and degenerative joint disease

The study describes the distribution of canine leucocyte antigens in synovial membrane biopsies from six dogs with canine rheumatoid arthritis (CRA) and from eight dogs with osteoarthritis (OA) secondary to spontaneous rupture of the cranial cruciate ligament (CCL) (n = 5) or patellar luxation (n = 3). Synovial membranes from five dogs without evidence of joint lesions were used as control tissues. In the subsynovium of dogs with normal joints CD5+, CD4+, CD8+ and alpha beta TCR+ lymphocytes were present only in low numbers. With monoclonal antibody (mAb) to MHC class II antigen, either none or up to 20-30% of synovial lining cells were immunoreactive. Furthermore, scattered MHCII+ stromal cells were seen in the deeper subsynovial layer. In synovial membrane biopsies from dogs with CRA numerous diffusely and perivascularly distributed CD5+ lymphocytes were found in the subsynovium. CD4+ cells outnumbered CD8+ cells and were more numerous in the perivascular areas. In all the CRA cases examined, there were markedly higher numbers of alpha beta TCR+ cells compared with gamma delta TCR+ cells. With mAb to CD21, low numbers of immunoreactive lymphocytes were demonstrated. In all the CRA cases, a marked increase of MHC class II antigen expression was noted. In the majority of samples, 50% or more than 90% of the synovial lining cells were strongly MHC class II+. Throughout the subsynovial layer there were numerous MHC class II+ cells and included those with dendritic morphology and inflammatory mononuclear cells. Furthermore, marked perivascular immunoreactivity for MHC class II antigen was found. In biopsies from dogs with OA, there were markedly lower numbers of subsynovial CD5+, CD4+ and CD8+ lymphocytes. T-cells were mainly diffusely distributed. In three of the eight OA dogs examined, there was an increased percentage of synovial lining cells expressing MHC class II. The majority of OA cases had subsynovial major histocompatibility complex (MHC) class II+ cells with a dendritic morphology.     

Anti-inflammatory effects of topical supernatant from human amniotic membrane cell culture on canine deep corneal ulcer after human amniotic membrane transplantation

The objective of this study was to examine the effect of topically applied human amniotic epithelial cell (HAEC) culture supernatant on corneal inflammatory reaction in dogs. Twenty-five dogs were randomly assigned into five groups. The control group consisted of five dogs with normal cornea. Inductions of corneal ulcers were performed using 0.45 cm trephine and human amniotic membrane was transplanted in 20 dogs. These 20 dogs were assigned into four treatment groups: topical antibiotic, topical corticosteroid, topical mock media and topical culture supernatant from HAEC, respectively. Administrations of the testing agents started at 24 h (h) after transplantation four times daily for nine consecutive days. Tears were collected before an operation 24 h after transplantation, but before application of the testing agents on consecutive odd days following transplantation. The concentrations of interleukin-1β (IL-1β) and nitric oxide (NO) in tear fluid were measured using canine IL-1β ELISA kit and Griess assay, respectively. Our analysis indicates that elevations of IL-1β and NO concentrations are associated with inflammatory conditions in the eyes. Corticosteroid, a reference anti-inflammatory drug, and the culture supernatant from HAEC significantly decreased IL-1β and NO concentrations. In addition, the clinical signs such as conjunctivitis and neovascularization were decreased in both topical corticosteroid and supernatant from HAEC treated groups. Mock and antibiotic solutions failed to decrease NO and IL-1β concentrations. In conclusion, topical application of the culture supernatant from HAEC alleviated inflammation in induced-corneal ulcer of dogs, possibly via inhibition of IL-1β and NO production.