Serum and synovial fluid concentrations of keratan sulfate and hyaluronan in dogs with induced stifle joint osteoarthritis following cranial cruciate ligament transection

OBJECTIVE: To examine longitudinal changes in serum and synovial fluid concentrations of keratan sulfate (KS) and hyaluronan (HA) after cranial cruciate ligament (CCL) transection in dogs. ANIMALS: 12 clinically normal adult mixed-breed dogs. PROCEDURE: Following CCL transection in the right stifle joint, KS and HA concentrations were determined in serum and neat (undiluted) synovial fluid prior to and 1, 2, 3, and 12 months after surgery. Postsurgical dilution of synovial fluid was corrected by use of urea as a passive marker. RESULTS: Synovial fluid KS and HA concentrations decreased at 1, 2, and 3 months after surgery in operated stifle joints, compared with baseline values. Synovial fluid KS concentration decreased in unoperated stifle joints at 1 month. A decrease in synovial fluid KS concentration was found in operated stifle joints, compared with unoperated stifle joints, at 2 and 3 months, and a decrease in synovial fluid HA concentrations was also found in operated stifle joints, compared with unoperated stifle joints, at 1, 2, and 3 months. Serum KS concentrations increased from baseline values at 3 months after surgery. Hyaluronan concentrations in operated stifle joints were lower than baseline values at 1, 2, and 3 months. Urea-adjusted synovial fluid concentrations revealed that dilution did not account for the decline in biomarker concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: The initial decrease and subsequent increase in synovial fluid concentrations of HA and KS may be caused by an acute inflammatory response to surgical intervention that negatively affects cartilage metabolism or an increase in production of immature proteoglycans.     

Apoptosis of ligamentous cells of the cranial cruciate ligament from stable stifle joints of dogs with partial cranial cruciate ligament rupture

OBJECTIVE: To describe the presence and amount of apoptotic ligamentous cells in different areas of partially ruptured canine cranial cruciate ligaments (prCCLs) and to compare these findings with apoptosis of ligamentous cells in totally ruptured cranial cruciate ligaments (trCCLs). ANIMALS: 20 dogs with prCCLs and 14 dogs with trCCLs. PROCEDURES: Dogs with prCCLs or trCCLs were admitted to the veterinary hospital for stifle joint treatment. Biopsy specimens of the intact area of prCCLs (group A) and the ruptured area of prCCLs (group B) as well as specimens from trCCLs (group C) were harvested during arthroscopy. Caspase-3 and poly (ADP-ribose) polymerase (PARP) detection were used to detect apoptotic ligamentous cells by immunohistochemistry. RESULTS: No difference was found in the degree of synovitis or osteophytosis between prCCLs and trCCLs. No difference was found in degenerative changes in ligaments between groups A and B. A substantial amount of apoptotic cells could be found in > 90% of all stained slides. A correlation (r(s) = 0.71) was found between the number of caspase-3-and PARP-positive cells. No significant difference was found in the amount of apoptotic cells among the 3 groups. No significant correlation could be detected between the degree of synovitis and apoptotic cells or osteophyte production and apoptotic cells. CONCLUSIONS AND CLINICAL RELEVANCE: The lack of difference between the 3 groups indicates that apoptosis could be a factor in the internal disease process leading to CCL rupture and is not primarily a consequence of the acute rupture of the ligament.     

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.     

Observer variability of tibial plateau slope measurement in 40 dogs with cranial cruciate ligament-deficient stifle joints

OBJECTIVE: To determine (1) the inter- and intraobserver variability in measurement of tibial plateau angle (TPA), (2) whether this inter- and intraobserver variability is related to the characteristics of the dog (age, size, and amount of degenerative joint disease [DJD]) and the experience level of the observer, and (3) the extent of any relationship between interobserver variability of TPA and the variability of the observers' selection of the specific cranial and caudal points along the tibial plateau. STUDY DESIGN: Examination of tibial radiographs of 40 dogs clinically affected with a cranial cruciate ligament (CrCL)-deficient stifle joint. METHODS: Eleven different observers, divided into 3 groups based on their level of experience with the tibial plateau leveling osteotomy (TPLO) technique, measured the TPA on all 40 radiographs on 5 different occasions. The degree of DJD present in the stifle joint was independently graded as an overall measure and then again as it specifically related to the cranial and caudal points along the tibial plateau. The total observed variabilities of the TPA were assessed with reference to interobserver differences, intraobserver differences, and among the groups of observers with respect to the different dog characteristics. Finally, the specific points selected on the radiographs were reexamined to determine whether any variability was present in cranial and caudal point selection. RESULTS: The interobserver standard deviation of the TPA measurements for each dog was 0.8 degrees, and the intraobserver standard deviation was 1.5 degrees. The TPA measurements obtained by the 11 observers differed significantly from each other (P <.001); however, there was no significant difference of TPA among the different groups of observers (P =.67). There was no significant correlation observed between either the inter- or intraobserver variability and the dog characteristics. Specific point data and their relationship to the various variables of dog characteristics and inter- and intraobserver TPA variability revealed significant correlations only to the amount of DJD present at the caudal point (P =.001). CONCLUSIONS: Interobserver variation, but no significant group variation, was present. Overall DJD did not appear to be related to the variability in TPA angle measurement. Most of the interobserver variability was attributable to variability in horizontal point selection at both the cranial and caudal points and vertical point selection at the caudal point. It appears that degenerative changes that specifically obscure the points on the tibial plateau, especially at the caudal point, are responsible for most of the interobserver variation. CLINICAL RELEVANCE: The desired postoperative TPA of 5 degrees is dependent on a precise initial measure of TPA preoperatively. This study indicates that there is statistically significant interobserver variability with measurement of TPA, which, therefore, can result in a similar amount of variability with the final tibial plateau slope obtained postoperatively.     

Synovitis in dogs with stable stifle joints and incipient cranial cruciate ligament rupture: a cross-sectional study

Objective: To evaluate stifle joints of dogs for synovitis, before development of joint instability and cranial cruciate ligament rupture (CrCLR). Study Design: Cross‐sectional study. Animals: Dogs (n=16) with CrCLR and stable contralateral stifles; 10 control dogs with intact CrCL. Methods: Arthritis and tibial translation were graded radiographically. Synovitis severity and cruciate pathology were assessed arthroscopically. Presence of inflammatory cells in synovial membrane biopsies was scored histologically. CrCLR stifle pairs and control stifles were compared. Results: Radiographic evidence of arthritis, cranial tibial translation, and arthroscopic synovitis were increased in unstable stifles, when compared with stable contralateral stifles in CrCLR dogs (P<.05). Arthroscopic synovitis in both joints of CrCLR dogs was increased compared with controls, was correlated with radiographic arthritis (S_R=0.71, P<.05), and was present in all stable contralateral stifles. Arthroscopically, 75% of stable stifle joints had CrCL fiber disruption, which correlated with severity of synovitis (S_R=0.56, P<.05). Histologic evidence of synovitis was identified in all CrCLR dogs, but was only significantly correlated with arthroscopic observations in stable stifles (r²=0.57, P<.005). Conclusion: Synovitis is an early feature of the CrCLR arthropathy in dogs before development of joint instability clinically. Severity of synovitis is correlated with radiographic arthritis in joints with minimal to no clinically detectable CrCL damage.     

Radiographic measurement of craniocaudal instability in stifle joints of clinically normal dogs and dogs with injury of a cranial cruciate ligament

OBJECTIVE: To determine craniocaudal laxity of the stifle joint of dogs when joints were positioned in tibial compression or neutral position. SAMPLE POPULATION: 19 normal stifle joints in 10 clinically normal dogs, 29 stifle joints with varying injury to the cranial cruciate ligament (10 complete ruptures alone, 10 complete ruptures with concomitant damage to the medial meniscus, 6 partial ruptures alone, and 3 partial ruptures with concomitant meniscal tearing), and 19 unaffected contralateral stifle joints in those 29 dogs. PROCEDURE: Relative displacement of bony landmarks was measured on paired lateral radiographs (neutral and tibial compression positions). Two measuring techniques were customized for use in dogs. RESULTS: The first technique failed to distinguish results in normal stifle joints from those in stifle joints with partial deficiency of cranial cruciate ligaments. Significant differences were found for joints with complete rupture, compared with stifle joints in clinically normal dogs. The second technique detected differences between normal stifle joints and injured joints with partial or complete rupture of the cranial cruciate ligament. Significant differences were not detected between joints with partial versus complete rupture. Adjusting data to account for size of dog did not improve results. CONCLUSIONS AND CLINICAL RELEVANCE: A wide range in measurements of laxity was found for stifle joints with intact cranial cruciate ligaments. Differences in degree of damage to the ligament and medial meniscus cannot be deduced from the amount of relative displacement measured on radiographs. Pathologic changes to the cranial cruciate ligament will not necessarily induce detectable changes in laxity of stifle joints in dogs.     

Expression of immune response genes in the stifle joint of dogs with oligoarthritis and degenerative cranial cruciate ligament rupture

Dysregulation of immune responses within joints plays an important role in development of inflammatory arthritis. We determined expression of a panel of immune response and matrix turnover genes in synovial fluid collected from a group of dogs with stifle oligoarthritis and associated degenerative cranial cruciate ligament (CCL) rupture (n=27). We also studied synovial fluid gene expression in dogs affected with other forms of degenerative arthritis (n=9) and in the stifle joint of healthy dogs with intact CCL (n=14). After collection, synovial cells were pelleted and RNA was isolated. Relative expression of cathepsin K, cathepsin S, tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), invariant chain (li), toll-like receptor-2 (TLR-2), and TLR-9 was determined using real-time quantitative RT-PCR. Data were normalized to peripheral blood mononuclear cells (PBMC) as an internal control. Relative expression of cathepsin K, MMP-9, TRAP, and li was increased in the stifle synovial fluid of dogs with oligoarthritis, when compared with the stifles of healthy dogs (P<0.05). In contrast, relative expression of all of the genes-of-interest in synovial fluid from joints affected with other forms of arthritis was not significantly different from the stifles of healthy dogs. TRAP expression was also significantly increased in the stifle joints of dogs with oligoarthritis, when compared to joint expression of TRAP in dogs with other forms of degenerative arthritis (P<0.05). In the dogs with stifle oligoarthritis, expression of both matrix turnover and immune response genes was increased in stifle synovial fluid, when compared with the internal PBMC control, whereas in healthy dogs and dogs with other forms of arthritis, only expression of matrix turnover genes was increased in synovial fluid, when compared with the internal PBMC control (P<0.05). Taken together, these findings suggest that antigen-specific immune responses within the stifle joint may be involved in the pathogenesis of persistent synovitis and associated joint degradation in dogs with oligoarthritis and degenerative CCL rupture.     

Absolute and relative cell counts for synovial fluid from clinically normal shoulder and stifle joints in cats

OBJECTIVE: To determine absolute and relative cell counts for synovial fluid from grossly, radiographically, and histologically normal shoulder and stifle joints in healthy cats. DESIGN: Clinical study. ANIMALS: 52 cats scheduled to be euthanatized for unrelated reasons. PROCEDURE: Arthrocentesis of the shoulder and stifle joints was performed bilaterally, and synovial fluid was analyzed for absolute WBC count, WBC morphology, and percentages of neutrophils and mononuclear cells. Joints were examined grossly and radiographically, and synovial membrane specimens were submitted for histologic examination. Synovial fluid samples that were contaminated with blood and samples from joints with any gross, radiographic, or histologic abnormalities were excluded. RESULTS: 82 of the 208 synovial fluid samples were excluded because abnormalities were identified during physical examination; the volume of fluid obtained was insufficient for analysis; there was evidence of blood contamination; or the joint had gross, radiographic, or histologic abnormalities. Median WBC count for the remaining 126 synovial fluid samples was 91 cells/microL (96.4% mononuclear cells and 3.6% neutrophils); WBC count was not significantly different between left and right joint samples or between shoulder and stifle joint samples. Body weight was associated with synovial fluid WBC count, with WBC count increasing as body weight increased. Sixteen of the 52 (30%) cats had radiographic evidence of osteoarthritis involving at least 1 joint. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that synovial fluid can be obtained reliably from shoulder and stifle joints in cats.     

Radiographic assessment of the progression of osteoarthrosis in the contralateral stifle joint of dogs with a ruptured cranial cruciate ligament

The formation and progression of osteoarthrosis in the unaffected contralateral stifle joints of 14 dogs with a unilateral cranial cruciate ligament rupture were monitored radiographically in terms of a global score and the scores for 10 parameters specific for the stifle joint. The dogs were examined initially and six and 12 months later by three observers, and the variability between the observers' scores was also assessed. The score for osteophytes at the tibial attachment site of the ligament was the most reliable parameter, and that for the increase in femoropatellar joint space was the least reliable. In the contralateral stifle joints there were significant increases after six and 12 months in osteophyte formation caudal to the tibial plateau, and in subchondral sclerosis of the tibial plateau and of the long digital extensor muscle groove. These three parameters progressed more regularly during the disease process than the other parameters. The global osteoarthrosis score of the contralateral stifle joint was an important risk factor for sustaining a rupture of the cranial cruciate ligament in that joint during the next six months.     

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.     

Detection of synovial macrophages in the joint capsule of dogs with naturally occurring rupture of the cranial cruciate ligament

OBJECTIVE: To test the hypotheses that the densities of macrophages in the synovial membranes and capsules of stifle joints in dogs with ruptured cranial cruciate ligaments are greater than those of normal joints and that those densities in affected joints are positively correlated with the chronicity and severity of the disease. ANIMALS: 17 dogs with naturally occurring rupture of the cranial cruciate ligament and 5 healthy control dogs. PROCEDURE: All dogs underwent orthopedic and radiographic evaluations. In affected dogs, duration of clinical signs was used as an indicator of disease chronicity and the severity of osteoarthritis in the stifle joint was determined radiographically. Joint capsule specimens were evaluated histologically; macrophages, interleukin-6, and tumor necrosis factor-alpha were identified by use of immunocytochemical techniques. RESULTS: Compared with unaffected joints, macrophage density was increased in all affected joints. Duration of disease was significantly associated with radiographic severity of osteoarthritis and synovial macrophage density. Synovial macrophage density was significantly associated with severity of osteoarthritis and with the presence of interleukin-6 and tumor necrosis factor-a. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that synovial macrophages may be involved in the development of pathologic changes (including osteophyte formation) in the stifle joints of dogs with osteoarthritis secondary to rupture of the cranial cruciate ligament. Determination of the importance of synovial macrophages in the development of changes in osteoarthritic joints may result in new treatment strategies that involve elimination of the deleterious effects of those cells.     

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.     

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.     

Osteophyte formation in the canine stifle joint following treatment for rupture of the cranial cruciate ligament

A clinical and radiological study of osteophyte formation following surgical or conservative treatment for rupture of the cranial cruciate ligament (C.C.L.), was carried out in thirty-eight dogs. Osteophyte formation had occurred in all of the affected stifle joints, in the period between treatment and the re-examination, irrespective of the method of treatment. In general, the degree of formation was less than that which had occurred before treatment. In the larger and more obese dogs, osteophyte formation was more extensive. The degree of lameness did not appear to be related to the degree of osteophyte formation radiographically evident at re-examination. No direct correlation could be established between the degree of joint instability, assessed at the re-examination, the degree of osteophyte formation following treatment, or the function of the limb.