Evaluation of ligament fibroblast viability in ruptured cranial cruciate ligament of dogs

OBJECTIVE: To determine fibroblast viability, assess development of apoptosis, and evaluate tissue hypoxia via histochemical, in-situ hybridization, or immunohistochemical staining in ruptured and intact cranial cruciate ligaments (CCLs) of dogs. ANIMALS: 32 dogs with ruptured CCLs, and 8 aged and 19 young dogs with intact CCLs. PROCEDURE: Markers of cell viability (lactate dehydrogenase [LDH]), apoptosis (terminal deoxynucleatidyl transferase-mediated deoxyuridine triphosphate-nick end labeling [TUNEL] method), and hypoxia (hypoxia-inducible factor-1alpha [HIF-1alpha] monoclonal antibody) were applied to CCL specimens; positive cells were assessed objectively (LDH) and subjectively (TUNEL and HIF-1alpha) in the main axial tissue component (core) and synovial intima and subintima (epiligamentous tissue). RESULTS: Viable fibroblasts were seen in all intact and ruptured CCLs. More nonviable cells were found in the core regions of ruptured CCLs and intact CCLs of young dogs than in the epiligamentous regions. Number of nonviable cells in the core region of ruptured CCLs was greater than that in intact CCLs of young and aged dogs, whereas the number in the epiligamentous region was similar in all specimens. The TUNEL and HIF-1alpha staining was only found in the epiligamentous region of ruptured CCLs. CONCLUSIONS AND CLINICAL RELEVANCE: Ruptured CCLs contained a high number of nonviable cells but not a great number of apoptotic cells. Repair processes in the epiligamentous region of the CCL include a metabolic response to hypoxia, suggesting that necrosis of ligament fibroblasts and transformation of surviving cells to a spheroid phenotype may be a response to hypoxia cause by microinjury or inadequate blood flow.     

Caudal cruciate ligament damage in dogs with cranial cruciate ligament rupture

Objective: To investigate the incidence of caudal cruciate ligament (CaCL) damage in dogs with cranial cruciate ligament rupture (CCLR). Study Design: Prospective clinical study. Animals: Dogs (n=24) admitted for surgical stabilization of the stifle after CCLR and 8 healthy dogs with intact cranial cruciate ligament (CCL) and CaCL studied as controls. Methods: Preoperative radiographs and stifle joint images (arthrotomy, 6; arthroscopy, 18) were collected from dogs with CCLR. Severity of arthritis, synovitis, CCL damage, and CaCL damage were assessed using numerical rating scales. The CaCL was probed to determine whether minor fraying or a full thickness defect in the ligament was present. Data collected from the study population were compared with the control population of dogs. Results: The CaCL was damaged in 21/24 (88%) of dogs with CCLR; 6/24 (25%) had a full thickness defect in the CaCL. Severity of stifle synovitis and severity of damage to the CaCL were positively correlated (P<.05). Conclusions: The CaCL is damaged in a high percentage of dogs with CCLR. A significant and positive correlation exists between the degree of synovitis present and the extent of CaCL damage. Clinical Relevance: In dogs with CCLR, cruciate ligament pathology typically involves both the CCL and CaCL. As the severity of synovitis and the extent of CaCL damage are related, this observation supports the hypothesis that stifle synovitis may contribute to CCL and CaCL degeneration and subsequent damage.     

Comparison of tibial plateau angles in dogs with and without cranial cruciate ligament injuries

OBJECTIVE: To measure and compare tibial plateau angles (TPA) of dogs with cranial cruciate ligament (CrCL) injuries and dogs without CrCL injuries. DESIGN: Prospective study. ANIMALS: 87 dogs. PROCEDURE: Stifle joints were measured from lateral radiographic views to determine TPA in 3 groups: group-1 dogs had CrCL injuries, group-1a dogs, a subgroup of group 1, had 1 unaffected stifle joint, and group-2 dogs had no CrCL injuries. Age, sex, breed, body weight, limb injured, and TPA were recorded for each dog. RESULTS: 56 stifle joints were measured in group-1 dogs; mean TPA was 23.76 degrees , and mean age and weight were 5.7 years and 37.91 kg (83.4 lb), respectively. Fourteen stifle joints were measured in group-1a dogs; mean TPA was 24.71 degrees , and mean age and weight were 5.6 years and 38.06 kg (83.8 lb), respectively. Sixty stifle joints were measured in group-2 dogs; mean TPA was 18.10 degrees , and mean age and weight of these dogs were 4.83 years and 35.85 kg (79 lb), respectively. The most common breeds included Labrador Retriever, Golden Retriever, and Rottweiler. The TPA of dogs in group 1 and group 1a were significantly greater than the TPA of dogs in group 2. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with CrCL injuries have a significantly greater TPA than dogs without CrCL injury. With further investigation, a normal TPA can be determined. In the future, TPA measurements may be used to screen dogs suspected of being susceptible to CrCL injury.     

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.     

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.     

Partial rupture of the cranial cruciate ligament in dogs

Four cases of partial rupture of the craniomedial part of the cranial cruciate ligament (CCL) are presented. Clinical examination revealed only subtle signs of CCL injury. The cranial drawer sign was present in two dogs and in flexion only. As the cranial drawer sign is not always evident a tentative diagnosis of partial CCL rupture should be based on history, joint tenderness and joint effusion. Arthrotomy and careful probing of the ligament is indicated. In these cases the lesion was treated immediately after diagnosis to prevent further degeneration and possible total rupture of the ligament. A fascial graft using the ‘over the top’ reconstruction technique was performed leaving the intact portion of the ligament in situ. Follow-up examination after four to six months revealed normal limb function in three dogs whereas slight and periodic lameness persisted in one dog.     

Immunopathological mechanisms in dogs with rupture of the cranial cruciate ligament

The majority of studies on cranial cruciate ligament (CrCL) disease to date have been carried out on dogs that already sustained a CrCL rupture, which is the end-stage of the disease. Investigations have recently been carried out to study humoral and cellular immunopathological mechanisms in predisposed dogs before clinical rupture of the contralateral CrCL. The cruciate ligaments are mainly composed of collagen type I, and immune responses to collagen have been suggested as a cause of CrCL degradation in dogs. None of these investigations showed evidence that anticollagen type I antibodies alone initiate CrCL damage. However, in predisposed dogs a distinct anticollagen type I antibody gradient was found towards the contralateral stifle joint that eventually sustained a CrCL rupture, suggesting that there was an inflammatory process present in these joints before detectable joint instability occurred. The importance of cellular reactivity to collagen type I in cruciate disease also remains unclear. Peripheral blood mononuclear cell proliferation to collagen type I was very diverse in dogs with cruciate disease whereas some sham operated dogs and healthy dogs tested positive as well. It is not yet determined whether cellular reactivity to collagen type I exists locally in the stifle joints nor whether this could initiate CrCL degradation. Inflammatory processes within the stifle joint can alter the composition of the cruciate ligaments. In animal models of immune-mediated synovitis, the mechanical strength of the CrCL is significantly reduced. Immunohistochemical studies on synovial tissues from dogs with rheumatoid arthritis and dogs with cruciate disease revealed that the pathologic features are similar in both joint pathologies and that the differences are mainly quantitative. Joint inflammation induced by biochemical factors such as cytokines has been implied in CrCL degeneration. In several studies, the levels of pro-inflammatory and T helper cytokines were measured in dogs that sustained a CrCL rupture, but the exact role of the various cytokines in the pathogenesis of CrCL disease remains inconclusive. More recently, the levels of the cytokines have been investigated over time in predisposed dogs before and after CrCL rupture. IL-8 expression tended to be higher in stifle joints that will rupture their CrCL during the next 6 months than in those that will not, indicating an inflammatory process in these joints before clinical rupture. This review provides a comprehensive overview of all possible implications of humoral and cell-mediated immune responses published in dogs with cruciate disease together with publications from human joint diseases. Furthermore, this review highlights recent findings on cytokines and proteinases in the accompanying joint inflammation.     

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.     

Tibial plateau leveling osteotomy in a llama with a ruptured cranial cruciate ligament

A 3-year-old 155-kg (342-lb) castrated male llama was examined because of left hind limb lameness of acute onset. A diagnosis of cranial cruciate ligament and medial collateral ligament rupture was made, and tibial plateau leveling osteotomy was recommended. The tibial plateau leveling osteotomy procedure was performed as described for dogs, except that 2 orthopedic plates were used to stabilize the osteotomy because of the size of the llama. The medial collateral ligament was sutured and reinforced with 2 strands of size-2 polypropylene placed in a figure-8 fashion between cancellous bone screws in the femur and tibia. Four days after surgery, failure of the medial collateral ligament repair was evident. Approximately 3.5 years after surgery, the llama was reexamined. The owners reported that the llama had full use of its left hind limb, and only mild lameness (grade 1 of 5) was evident. Results suggest that tibial plateau leveling osteotomy may be applicable in camelids with rupture of the cranial cruciate ligament. However, additional study is needed before tibial plateau leveling osteotomy can be routinely recommended. In particular, additional information is needed on the tibial plateau slope in healthy camelids, the role of the fibula in tibial plateau leveling osteotomy procedures, and the prevalence of cranial cruciate ligament rupture in camelids.     

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.     

Long-term outcome of surgery for dogs with cranial cruciate ligament deficiency

Fifty-eight dogs with cranial cruciate ligament deficiency were assessed and treated surgically. At an average of 50 months postoperatively, the functional outcome was assessed by means of an owner-based clinical assessment and a clinical examination. Client-based data were available for 26 dogs and 20 dogs were reassessed after 50 months. The results were compared with the initial values and with data from an assessment 13 months postoperatively. The level of disability at 50 months was judged to be significantly less than initially. However, there were no differences between the initial assessments and those made after 50 months for the perceived 'effect of cold weather' and the dogs' 'ability to jump', despite both measures having improved after 13 months. Age and meniscal injury were identified as poor prognostic indicators for the long-term outcome. The equivalent joint on the contralateral limb deteriorated significantly during the study.     

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.     

Assessing the efficacy of perioperative carprofen administration in dogs undergoing surgical repair of a ruptured cranial cruciate ligament

Twenty-one otherwise healthy dogs that presented for surgical repair of a ruptured cranial cruciate ligament were blindly and randomly given either carprofen (2.2 mg/kg body weight, orally) or a placebo beginning 12 hours preoperatively and continuing every 12 hours for a total of three doses. The patients were assessed for postoperative pain using a subjective pain score and given oxymorphone (0.1 mg/kg body weight, intramuscularly) every four hours if the pain score was 2 or greater. Blood samples were also collected to determine serum cortisol levels. There was a significant increase in serum cortisol levels in the immediate postoperative period in both the placebo group and the carprofen group (p less than 0.05). There was no significant difference in the percentage of increase in serum cortisol levels between the two groups. No correlation was evident between the serum cortisol levels and the corresponding pain scores in either group. This subjective method of assessing postoperative pain was not accurate and should not be relied upon for determination of postoperative analgesic administration. Perioperative oral administration of carprofen did not appear to be effective in controlling postoperative pain in these patients.     

Changes in concentrations of biochemical markers of osteoarthritis following surgical repair of ruptured cranial cruciate ligaments in dogs.

OBJECTIVE: To investigate longitudinal changes in concentrations of the 1/20/5D4 epitope (5D4) of keratan sulfate and total sulfated glycosaminoglycans (S-GAG) in synovial fluid and serum of dogs with cranial cruciate ligament (CCL) rupture that was repaired via intra-articular surgery. ANIMALS: 58 dogs with a ruptured CCL and osteoarthritis of the affected (index) joint. PROCEDURE: Prior to surgical repair of the ruptured CCL, 5D4 concentration was measured in serum and synovial fluid samples by use of an inhibition ELISA, and total S-GAG concentration was measured in synovial fluid samples by use of a direct dye-binding assay. Ruptured CCL were repaired surgically, using an intra-articular fascial graft. Dogs were reexamined 1.5, 7, and 13 months after surgery, and 5D4 and S-GAG concentrations in synovial fluid and serum were measured again. RESULTS: Serum 5D4 concentrations did not change significantly during the study. Concentrations of 5D4 in synovial fluid (expressed as a ratio of S-GAG concentration) did change significantly with time. In the index joint, the 5D4:S-GAG decreased from 0.19 at the beginning of the study to 0.09 1.5 months after surgery, but 7 months after surgery, the ratio increased again to 0.20. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the hypothesis that serum concentration of 5D4 is not a useful marker of osteoarthritis in dogs. Surgical intervention transiently reduced the concentration of 5D4 in synovial fluid but had no effect on S-GAG concentration.