Expression of interleukin-8 and intercellular cell adhesion molecule-1 in the synovial membrane and cranial cruciate ligament of dogs after rupture of the ligament

This cross-sectional clinical study compared inflammation, including expression of the chemokine interleukin (IL)-8 and intercellular cell adhesion molecule-1 (ICAM-1), in the stifle joints of 4 control dogs and 23 dogs with cranial cruciate ligament rupture (CCLR). The CCL, synovial membrane, meniscus, cartilage, and synovial fluid from the affected stifle joints of all the dogs were examined. Inflammatory cell counts were performed on the synovial fluid, and the tissues were processed for histologic study and immunohistochemical detection of IL-8 and ICAM-1. The synovial fluid from the stifle joints of the dogs with CCLR had an increased percentage of neutrophils (P = 0.054) and a decreased percentage of lymphocytes (P = 0.004) but not macrophages compared with the fluid from the control dogs. There was accumulation of inflammatory cells and increased expression of IL-8 and ICAM-1 in the vascular endothelium of the synovial membrane and the CCL of the dogs with CCLR. The increase in inflammatory cells in the stifle joints of dogs with CCLR may therefore be due to increased expression of IL-8 and ICAM-1 in the synovial membrane and the CCL after the injury. These data may help in understanding the mechanisms of inflammation associated with CCLR.     

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.     

Degenerative changes of the cranial cruciate ligament harvested from dogs with cranial cruciate ligament rupture

Degenerative cranial cruciate ligament (CCL) rupture is characterized histologically by degenerating extracellular matrix (ECM) and chondroid metaplasia. Here, we describe the progression of chondroid metaplasia and the changes in the expression of ECM components in canine CCL rupture (CCLR). CCLs from 26 stifle joints with CCLR (CCLR group) and normal CCLs from 12 young beagles (control group) were examined histologically and immunohistochemically for expression of type I (COLI), type II (COLII), type III collagen (COLIII) and Sry-type HMG box 9 (SOX9). Cell density and morphology of CCLs were quantified using hematoxylin–eosin staining. The percentage of round cells was higher in the CCLR group than in controls. COLI-positive areas were seen extensively in the connecting fibers, but weakly represented in the cytoplasm of normal CCLs. In the CCLR group, there were fewer COLI-positive areas, but many COLI-positive cells. The percentages of COLII-, COLIII- and SOX9-positive cells were higher in the CCLR group than in controls. The number of spindle cells with perinuclear halo was high in the CCLR group, and most of these cells were SOX9-positive. Deposition of COLI, the main ECM component of ligaments, decreased with increased COLIII expression in degenerated CCL tissue, which shows that the deposition of the ECM is changed in CCLR. On the contrary, expression of SOX9 increased, which may contribute to the synthesis of cartilage matrix. The expression of COLII and SOX9 in ligamentocytes showed that these cells tend to differentiate into chondrocytes.     

Are bacterial load and synovitis related in dogs with inflammatory stifle arthritis?

It has been proposed that small quantities of microbial material within synovial joints may act as a trigger for development of synovitis. We have previously identified an association between intra-articular bacteria and development of inflammatory stifle arthritis and cranial cruciate ligament rupture (CCLR) in dogs, and now wished to quantify bacterial load and markers of synovitis in dogs with and without stifle arthritis and CCLR. Joint tissues were collected from dogs with CCLR (n=51) and healthy dogs with normal stifles (n=9). Arthritis was assessed radiographically in CCLR dogs. Bacterial load was assessed using qPCR and broad-ranging 16S rRNA primers. qRT-PCR was used to estimate expression of the T lymphocyte antigen receptor (TCR Vβ), CD3?, tartrate-resistant acid phosphatase (TRAP), IL-4, IL-17, and TNF-α genes. Severity of synovitis was assessed histologically. Bacterial load was increased in arthritic stifles, when compared with healthy stifles. Histologic synovitis in arthritic stifles was mononuclear and was significantly correlated with bacterial load (1 of 2 primer sets) (S(R)=0.49, p<0.001). In arthritic stifles, expression of TRAP in synovium was increased relative to healthy stifles. Expression of pro-inflammatory genes was not correlated with bacterial load, histologic inflammation, or radiographic arthritis. Translocation of bacterial material to the canine stifle is related to the presence of joint inflammation. The lack of a strong positive correlation suggests that bacterial load is unlikely to be a primary pro-inflammatory factor. However, dysregulation of immune responses within synovial tissues may be dependent upon an environmental microbial trigger.     

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.     

Cranial cruciate ligament injury in the dog: pathophysiology, diagnosis and treatment

Cranial cruciate ligament (CCL) disease in the dog is a multifactorial complex problem that requires a thorough understanding of the biomechanics of the stifle joint to be understood. Successful treatment of rupture of the CCL should be based on managing underlying anatomical and conformational abnormalities rather than attempting to eliminate the tibial cranial drawer sign. The cranial and caudal cruciate ligaments, the patella ligament and quadriceps mechanism, the medial and lateral collateral ligaments, the medial and lateral menisci and the joint capsule provide stability of the joint and load-sharing. The function of the stifle is also significantly influenced by the musculature of the pelvic limb. An active model of biomechanics of the stifle has been described that incorporates not only the ligamentous structures of the stifle but also the forces created by weight-bearing and the musculature of the pelvic limb. This model recognises a force called cranial tibial thrust, which occurs during weight-bearing, and causes compression of the femoral condyles against the tibial plateau. In middle-aged, large-breed dogs, forces acting on the CCL together with conformation-related mild hyperextension of the stifle and slightly increased tibial plateau slopes are suspected to cause progressive degeneration of the ligament. Palpation of craniolateral stifle laxity has become pathognomonic for CCL rupture; however, chronic periarticular fibrosis, a partial CCL rupture, and a tense patient, may make evaluation of instability of the stifle difficult. Surgical treatment is broadly separated into three groups: intracapsular, extracapsular, and tibial osteotomy techniques. Tibial osteotomy techniques do not serve to provide stability of the stifle but rather alter the geometry of the joint to eliminate cranial tibial thrust such that functional joint stability is achieved during weight-bearing. Visualisation of both menisci is a critical aspect of CCL surgery, irrespective of the technique being performed. Regardless of the surgical technique employed, approximately 85% of dogs show clinical improvement. However, many of these dogs will demonstrate intermittent pain or lameness. Post-operative management is an integral part of the treatment of CCL rupture, and significant benefits in limb function occur when formalised post-operative physiotherapy is performed.     

Prevalence of cranial cruciate ligament rupture in a population of dogs with lameness previously attributed to hip dysplasia: 369 cases (1994-2003)

OBJECTIVE: To determine the prevalence of cranial cruciate ligament rupture (CCLR) in dogs with lameness previously attributed to canine hip dysplasia (CHD). DESIGN: Retrospective study. ANIMALS: 369 client-owned dogs. PROCEDURES: Hospital medical records from 1994 to 2003 were reviewed for dogs in which the referring veterinarian had diagnosed hip dysplasia or hip pain. Dogs were designated as having hind limb lameness because of partial or complete CCLR or CHD. RESULTS: 8% of dogs were sexually intact females, 43% were spayed females, 14% were sexually intact males, and 35% were castrated males. Mean age was 3.8 years (range, 3 months to 15 years). The most common breeds were the Labrador Retriever (21%), German Shepherd Dog (13%), and Golden Retriever (11%). The prevalence of CCLR as the cause of hind limb lameness was 32% (95% confidence interval, 27.2% to 36.8%). The distribution of CCLR among hind limbs was left (29%), right (28%), and bilateral (43%). Of 119 dogs with CCLR, 94% had concurrent radiographic signs of CHD, 92% had stifle joint effusion, and 81% had a cranial drawer sign. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of the high prevalence of CCLR in dogs referred for lameness because of CHD, it is important to exclude other sources of stifle joint disease before making recommendations for treatment of CHD.     

Localization of cathepsin K and tartrate-resistant acid phosphatase in synovium and cranial cruciate ligament in dogs with cruciate disease

OBJECTIVE: To localize cathepsin K and tartrate-resistant acid phosphatase (TRAP) in synovium and cranial cruciate ligament (CCL) of dogs with cruciate disease. ANIMALS: Dogs (n=15) with cruciate disease and ruptured CCL, and 12 dogs with intact CCL. METHODS: Synovium and CCL were examined histologically and cells containing cathepsin K or TRAP were identified immunohistochemically and histochemically, respectively. RESULTS: Increased cellular localization of cathepsin K and TRAP was detected in synovium and ruptured CCL in dogs with cruciate disease, when compared with tissues from dogs with intact CCL. Inflammation of synovium with TRAP+ macrophage-like cells was seen in 73% of dogs with CCL disease, but was not seen in dogs with intact CCL. The presence of cathepsin K and TRAP protein in synovium and CCL tissues was significantly correlated in dogs with CCL rupture. CONCLUSION: Inflammation of the epiligament of ruptured CCL with cathepsin K+ and TRAP+ macrophage-like cells forms part of a similar, more generalized chronic inflammatory change within the periarticular tissues of the stifle of a large proportion of dogs with CCL rupture. CLINICAL RELEVANCE: Production of matrix-degrading enzymes by the synovium may induce progressive pathologic rupture of the CCL. Therefore, these collagenolytic pathways may offer a novel target for medical therapy of joint inflammation in canine patients with cruciate disease.     

Correlative biomechanical and histologic study of the cranial cruciate ligament in dogs

The mechanical properties of the cranial cruciate ligament were determined, using unilateral bone-ligament-bone preparations from 65 dogs of various ages and body sizes. Tensile loading of the cranial cruciate ligament from 1 of each pair of stifle joints demonstrated a decrease in material properties (modulus, maximum stress, strain energy) with aging. The decreases in maximum stress and strain energy with age were significantly less (P less than 0.05 and P less than 0.05, respectively) in the cranial cruciate ligament from dogs weighing less than 15 kg, compared with those weighing 15 kg or more. The cranial cruciate ligament and remaining femorotibial ligaments were collected from the opposite stifle joints and examined microscopically. By 5 years of age, the cranial cruciate ligaments of dogs weighing greater than 15 kg consistently had microscopic evidence of degenerative disease (loss of ligamentocytes, metaplasia of surviving ligamentocytes to chondrocytes, and failure to maintain collagen fibers and primary collagen bundles) which progressed in severity with age. The caudal cruciate ligaments were similarly affected, although the degenerative changes were rarely as severe as in the cranial cruciate ligament. The collateral ligaments underwent minimal degeneration. Sex differences had no bearing on degeneration. The cranial cruciate ligaments in dogs weighing less than 15 kg generally had less severe alterations than those in heavier dogs, and the onset of the degenerative process was delayed by several years. Cranial cruciate ligaments removed from dogs after ligament failure not only had degenerative disease, but also had undergone unsuccessful attempts at repair. In contrast, fibrous repair was rarely present in intact ligaments of asymptomatic dogs with degenerative disease of the cranial cruciate ligament.     

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.     

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.     

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.     

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.     

Morphologic and functional features of the canine cruciate ligaments

OBJECTIVE: To review the gross, microscopic, and functional anatomy of the cranial cruciate ligament (CCL) in dogs. STUDY DESIGN: Literature review. METHODS: Reports of the anatomy and function of the cruciate ligaments in dogs were retrieved by search of the 1975-2005 PubMed database. RESULTS: The CCL has an important biomechanical function resisting cranial drawer, hyperextension, and internal rotation and acts to fine tune and guide the stifle through its rolling and sliding motion. It has a complex architecture, and distinct geographic regions within the ligament have different functional roles depending on the angle and loading conditions. Collagen type I is the main component of the extracellular matrix; the fibrils have a crimped structure. The cruciate ligaments are almost completely covered by synovium, protecting them from synovial fluid. Cruciate blood supply is mainly of soft tissue origin. The intraligamentous network is relatively limited whereas the core of the middle third of the CCL is even less well vascularized. Neurohistologic studies are very limited in the dog. Various mechanoreceptors and proprioceptive receptors have been identified within the substance of the cruciate ligaments. CONCLUSIONS: CCL structural characteristics play an important part in its complex behaviour with the crimped pattern of the collagen fibrils being an important determinant of its biomechanical properties. In contrast to reports of managing CCL rupture, there are few reports describing the microanatomy and neurovascular morphology of the cruciate ligaments. CLINICAL RELEVANCE: Cruciate disease is likely multi-factorial. Improved understanding of CCL degradation leading to CCL rupture is critical to development of new diagnostic tests for cruciate disease in dogs. Appropriate intervention during the early stages of disease process might preserve CCL structural properties by preventing further collagen degradation. Accurate knowledge of functional and fiber bundle anatomy is imperative for reconstruction and restoration of normal stifle joint physiology. Reconstructive goals should alleviate existing instability and mimic normal kinematics. Knowledge of the exact function of the CCL in the neuromuscular control around the stifle joint could possibly explain osteoarthritis progression after CCL damage.     

In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk

Primary fibroblast cultures of canine cranial (CCL) and caudal (CaCL) cruciate ligaments were stimulated with different apoptosis inducers with or without preincubation of the pancaspase inhibitor zVAD.fmk. In contrast to CaCL fibroblasts, fibroblasts from CCL were significantly more susceptible to apoptosis inducers of the intrinsic pathway like doxorubicin, cisplatin and nitric oxide (NO)-donors and to Fas ligand (FasL), an apoptosis inducer of the death receptor pathway. Apoptotic response to staurosporine and the peroxynitrite donor GEA was similar in both ligament fibroblasts. Stimulation with dexamethasone or TNFα could not induce apoptosis in CCL and CaCL fibroblasts, in spite of present TNFR1 and TNFR2 receptors. zVAD.fmk was able to prevent apoptosis in up to 66% of CCL cells when treated with FasL, cisplatin or doxorubicin but it had no effect on NO or peroxynitrite induced apoptosis. In conclusion, differential susceptibility to apoptotic triggers like FasL or NO between cranial and caudal cruciate ligament fibroblasts in vitro may be a reflection of the different susceptibilities to degenerative rupture of the ligament. These findings indicate that a general caspase inhibition does not completely protect canine CCL cells from apoptosis.     

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.     

Lymphocyte Populations in Joint Tissues from Dogs with Inflammatory Stifle Arthritis and Associated Degenerative Cranial Cruciate Ligament Rupture

Objective: To evaluate lymphocyte populations in stifle synovium and synovial fluid of dogs with degenerative cranial cruciate ligament rupture (CCLR). Study Design: Prospective clinical study. Animals: Dogs (n=25) with stifle arthritis and CCLR, 7 dogs with arthritis associated with cartilage degeneration (osteoarthritis [OA]), and 12 healthy Beagle dogs with intact CCL. Methods: Arthritis was graded radiographically in CCLR dogs. After collection of joint tissues, mononuclear cells were isolated and subsequently analyzed using flow cytometry for expression of CD3, CD4, CD8, and CD21. Results: The proportions of CD4⁺ T helper lymphocytes, CD8⁺ cytotoxic T lymphocytes, and CD3⁺CD4^?CD8^? T lymphocytes were increased in synovium from dogs with CCLR compared with synovium from healthy Beagle dogs (P<.05). The proportion of CD3⁺CD4^?CD8^? T lymphocytes in synovial fluid was increased in dogs with CCLR compared with dogs with OA (P<.05). In dogs with CCLR, the proportion of CD3⁺CD4^?CD8^? T lymphocytes in synovial fluid was inversely correlated with radiographic arthritis (S_R=?0.68, P<.005). Conclusion: Lymphocytic inflammation of stifle synovium and synovial fluid is an important feature of the CCLR arthropathy. Lymphocyte populations include T lymphocytes expressing CD4 and CD8, and CD3⁺CD4^?CD8^? T lymphocytes. Presence of CD3⁺CD4^?CD8^? T lymphocytes was associated with development of stifle synovitis. Further work is needed to fully identify the phenotype of these cells.     

Proinflammatory cytokine activities, matrix metalloproteinase-3 activity, and sulfated glycosaminoglycan content in synovial fluid of dogs with naturally acquired cranial cruciate ligament rupture

OBJECTIVE: To measure and compare activities of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinase-3 (MMP-3); as well as sulfated glycosaminoglycan (S-GAG) content in synovial fluid from dogs with cranial cruciate ligament rupture (CCLR) and dogs with clinically normal stifles. To determine whether correlations exist between demographic and disease-related variables and these synovial markers. STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs with CCLR (n=23) and Beagles with normal stifle joints (n=21). METHODS: Synovial fluid activities of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) were determined by bioassay. MMP-3 activity was measured using fluorogenic substrate. S-GAG contents were determined by dimethylmethylene blue dye-binding assay. Mann-Whitney U-test was used to compare results from CCLR joints with normal controls. Spearman's rank correlation test was used to evaluate associations between demographic and disease-related markers and synovial markers. RESULTS: Mean values for synovial markers were significantly higher in CCLR joints compared with controls. IL-1beta and MMP-3 were positively correlated with lameness duration. CONCLUSIONS: Activities of proinflammatory cytokines, MMP-3 activity and S-GAG contents were significantly elevated in synovial fluid from canine stifle joints with naturally acquired CCLR. These results indicate that there is joint inflammation and increased release of GAGs into synovial fluid, suggesting that these inflammatory changes are associated with depletion of proteoglycan from articular cartilage. CLINICAL RELEVANCE: Medical and surgical treatments designed to decrease joint inflammation and breakdown of proteoglycans may be of value in the management of CCLR in the dog.     

COL-3 inhibition of collagen fragmentation in ruptured cranial cruciate ligament explants from dogs with stifle arthritis

Inhibition of collagen fragment generation in canine cranial cruciate ligament (CCL) explant cultures by the matrix metalloprotease inhibitor (6-demethyl)-6-deoxy-4-dedimethylamino tetracycline (COL-3) was studied. Cranial cruciate ligament specimens were collected from dogs with inflammatory stifle arthritis/CCL rupture and dogs with normal stifles. Explant cultures from each CCL specimen included one COL-3 treated explant and a baseline control; explants from 12 ruptured CCLs were prepared in triplicate and a protease inhibitor cocktail positive control was used. Explant supernatants were analyzed for generation of collagen fragments after two days. Treatment of ruptured CCL explants with 10(-4)M COL-3 decreased generation of collagen fragments. The extent of this inhibition was increased in explants treated with a protease inhibitor cocktail. Generation of collagen fragments was increased in ruptured CCLs, when compared with intact CCLs. It is concluded that generation of collagen fragments was increased in pathological ruptured CCL explants. This degradation could be significantly inhibited in vitro by 10(-4)M COL-3.