Generation and characterization of mesenchymal stem cells from the affected femoral heads of dogs with Legg Calvé Perthes disease.

Background: Canine Legg Calvé Perthes disease (LCPD) occurs during the growth period, and the cause of ischemic necrosis of the femoral head during growth remains unclear. If LCPD-affected femoral head-derived mesenchymal stem cells (LCPD-MSCs) can be generated, they can be used as a new tool for the pathophysiological analysis of canine LCPD. Aim: To generate affected femoral head-derived mesenchymal stem cells (MSCs) from dogs with LCPD and investigate the mRNA expression levels of angiogenesis-related factors and osteogenic differentiation potency of LCPD-MSCs. Methods: This study was performed using affected femoral heads from dogs diagnosed with LCPD and underwent femoral head and neck ostectomy. The necrotic tissue was harvested from the LCPD-affected femoral head and cultured statically (LCPD group, n = 6). Canine bone marrow-derived MSCs (BM-MSCs) were used as controls (control group, n = 6). First, the morphology of the cultured cells was observed, and the expression of CD29, CD34, CD44, CD45, CD90, and major histocompatibility complex class II was analyzed using flow cytometry. Additionally, the trilineage differentiation potency of the LCPD-affected head-derived adherent cells was examined. Furthermore, the expression levels of HIF1A, VEGFA, VEGFB, and PDGFB mRNAs and the bone differentiation potency of LCPD-affected head-derived adherent cells were investigated. Results: LCPD-affected femoral head-derived adherent cells showed a fibroblast-like morphology, and the expression of cell surface antigens was similar to that of BM-MSCs. In addition, LCPD-affected femoral head-derived adherent cells showed the same trilineage differentiation potency as BM-MSCs and were consistent with MSC characteristics. Furthermore, the mRNA expression levels of angiogenesis-related factors could be objectively measured in LCPD-MSCs and those MSCs had bone differentiation potency. Conclusion: In the present study, canine LCPD-MSCs were successfully generated, suggesting their usefulness as a tool for pathological analysis of LCPD in dogs.

A pilot study of microRNA assessment as a means to identify novel biomarkers of spontaneous osteoarthritis in dogs.

MicroRNAs (miRNAs) are important regulators of intercellular signaling and are promising biomarkers in osteoarthritis (OA). In this study, comprehensive analysis was performed to identify miRNAs involved in the pathogenesis of spontaneous OA in dogs. Dogs diagnosed with OA based on radiography and arthroscopy of the stifle joint were included in the OA group. Dogs without any evidence of orthopedic disease were included in the unaffected group. To investigate miRNA expression levels, RNA sequencing analysis (RNA-seq) was performed in synovial tissue (OA group: n = 3, Unaffected group: n = 3) and RT-qPCR was performed in synovial tissue, synovial fluid and serum (OA group: n = 17, Unaffected group: n = 6), and compared between the two groups. The RNA-seq results showed that 57 miRNAs were significantly upregulated and 42 were significantly downregulated in the OA group. Specifically, miR-542 and miR-543 expression levels in the synovial tissue, synovial fluid, and serum were consistently higher in the OA group than in the unaffected group, suggesting that these miRNAs may be used as biomarkers for detecting canine OA. This is the first report to comprehensively analyze the expression patterns of miRNAs in the synovial tissue of dogs with spontaneous OA.

Evaluation of Hindlimb Deformity and Posture in Dogs with Grade 2 Medial Patellar Luxation during Awake Computed Tomography Imaging while Standing.

OBJECTIVE: The aim of this study was to determine the degree of bone deformities and hindlimb postural abnormalities in a standing position in awake Toy poodles with and without grade 2 medial patellar luxation (MPL) using high speed 320-row computed tomography (CT). METHODS: The limbs with grade 2 MPL (MPL-G2 group) and without any orthopaedic disorders (control group) were imaged in a standing position, without sedation or anaesthesia, using CT. In MPL-G2 group, images were obtained when the patella was luxated (G2-L group) and reduced (non-luxation, G2-NL group). Bone morphologies of the femur and tibia were quantified three-dimensionally. Hindlimb standing posture was evaluated by measuring femoral rotation and abduction angles, tibial rotation angle, metatarsal rotation angle, foot rotation angle, angle between the femoral anatomical axis and the mechanical axis of hindlimb and stifle joint line convergence angle. RESULTS: There were no significant differences in bone morphologic parameters between the MPL-G2 group (5 limbs) and the control group (6 limbs). In the G2-NL group, there were no significant hindlimb postural abnormalities. In contrast, in the G2-L group, significant hindlimb postural abnormalities including external rotation of femur, internal rotation of tibia and foot, external rotation of tarsal joint, large stifle joint convergence angle, genu varum and toe-in standing were observed. CONCLUSION: Dogs with grade 2 MPL have no bone deformities but show abnormal standing posture when the patella is luxated.

Functional Anatomy of the Craniolateral and Caudomedial Bundles of the Caudal Cruciate Ligament in Beagle Dogs.

OBJECTIVE: The aim of this study was to investigate functional anatomy of the craniolateral bundle (CrLB) and caudomedial bundle (CdMB) of caudal cruciate ligament in normal Beagles. MATERIALS AND METHODS: Twelve stifle joints of 12 Beagle cadavers that were euthanatized for reasons other than musculoskeletal disease were used. Anatomical characteristics of femoral and tibial attachment regions of the caudal cruciate ligament were described, and location and area of the footprints of the CrLB and CdMB were objectively determined. The CrLB was artificially divided into two equal fibre bundle units and the CdMB was artificially divided into four equal bundle units. Changes in the fibre arrangement were recorded during stifle range of motion, and the tension of each fibre bundle unit at various stifle joint angles was objectively measured. RESULTS: The CrLB was attached to the intercondylar fossa of the femur, and the CdMB was attached to the medial surface of the medial condyle of the femur. The centre of the CrLB attachment site was located craniomedial to that of the CdMB on the tibia. During stifle range of motion, each divided fibre bundle unit in the CrLB and CdMB was twisted externally. The tension of entire CdMB tended to be higher than that of entire CrLB at 180 to 135 degrees of the stifle angles. In full extension angle, the tension of entire CdMB was 1.55 times higher than that of entire CrLB. CLINICAL SIGNIFICANCE: This study contributes to understanding of the biomechanics and the function of the caudal cruciate ligament, which help to elucidate the pathophysiology of caudal cruciate ligament tear and to develop stifle joint stabilization surgery.

Variations in gene expression levels with severity of synovitis in dogs with naturally occurring stifle osteoarthritis.

Osteoarthritis (OA) is one of the major causes of chronic pain in dogs. However, the pathogenesis of OA has not been fully understood in dogs. The objective of this study was to comprehensively investigate the mRNA expression levels of proinflammatory cytokines, inflammatory mediators, nerve growth factor and its receptor, and matrix metalloproteinases in the synovium of dogs with spontaneous OA as well as to elucidate their relationships with the severity of synovitis. Dogs that were diagnosed with stifle OA on the basis of radiographic findings were included, and the degree of synovitis was observed using stifle arthroscopy. The dogs were assigned to two different groups depending on their synovitis scores: the low-grade group (score of 1 or 2; n = 8) and high-grade group (score of 3 to 5; n = 18). The dogs showing no evidence of orthopedic disease were included in the control group (n = 6). Synovial tissue samples were collected from the sites at which synovitis scores were assessed using arthroscopy. Total RNA was extracted from the collected synovial tissue, and cDNA was synthesized. Subsequently, RT-qPCR were performed using canine-specific primer sets for IL1B, IL6, CXCL8, TNF, TGFB1, PTGS2, PTGES, MMP3, MMP13, NGF, NTRK1, and PTGER4. Expression levels of IL1B, IL6, CXCL8, and MMP13 were significantly higher in the high-grade group than in the control group. In addition, expression levels of IL1B, CXCL8, TNF, and PTGS2 were significantly higher in the high-grade group than in the low-grade group. Expression levels of IL1B, IL6, CXCL8, TNF, PTGS2, and PTGER4 showed significant positive correlation with synovitis score. In conclusion, all mRNA expression levels in the synovial membrane varied according to the degree of synovitis in dogs with spontaneous OA. Thus, this study may partially elucidate the pathogenesis of synovitis in dogs with spontaneous OA.

Treatment of methicillin-resistant Staphylococcus aureus infection following tibial plateau leveling osteotomy in a dog.

BACKGROUND: In recent years, surgical site infections caused by drug-resistant pathogens have emerged as a cause of concern in small animal practice. In this report, methicillin-resistant Staphylococcus aureus (MRSA) infections associated with tibial plateau leveling osteotomy (TPLO) is reported. However, there have been no reports on the treatment of MRSA infection following TPLO in dogs. This case report describes the use of a combination of vancomycin and rifampicin to treat MRSA infection following TPLO in a dog. CASE DESCRIPTION: A 7-year-old spayed female American cocker spaniel was referred for right hind limb lameness that did not improve with conservative treatment. The dog was diagnosed with cranial cruciate ligament rupture, for which TPLO was performed. Once the surgical wound was closed, the dog licked the skin on the surgical site, causing the injury to dehisce. MRSA was detected from the purulent discharge, and chloramphenicol was then administered based on the drug sensitivity test results. Because of the continued drainage, the implants were removed after the bone union of the osteotomy site was observed. Since this did not provide any relief to the existing condition, the antibiotic was changed to vancomycin at 132 days after TPLO surgery, and the infected location was cleaned many times through a drain tube placed into the tibia. However, the infection could not be controlled. Thus, a rifampicin and vancomycin combination was started. As a result, the purulent discharge disappeared and the fistula entirely closed on the 154th day after TPLO surgery. CONCLUSION: A combination of rifampicin and vancomycin may be effective for treating MRSA infection at the surgical site following TPLO surgery that does not heal despite implant removal and administration of vancomycin.

Utility of a novel activity monitor assessing physical activities and sleep quality in cats.

Osteoarthritis in cats is more prevalent because cats are living longer with advancement in veterinary medicine. Objective evaluation of behavioral changes in cats with osteoarthritis can facilitate an early diagnosis. The objective of this study was to investigate the utility of a novel activity monitor for analyzing physical activities and sleep quality in cats. First, a novel activity monitor (Plus Cycle®; JARMeC, Kanagawa, Japan), with a built-in three-directional accelerometer and an air pressure sensor specifically designed for cats, was compared to a human activity monitor (Actical®; Philips Respironics, OR, USA) previously used in cats (n = 10). Second, the validity of the measurement accuracy of the amount of physical activity, the number of vibrations, the number of jumps, and the resting and sleeping time was evaluated using Plus Cycle® in healthy cats (n = 6). Finally, the effects of gender and age of cats and time of day on the amount of physical activity, the number of vibrations, the number of jumps, and the resting and sleeping time were investigated in client-owned cats (n = 61). There were strong correlations between Plus Cycle® and Actical® in total activity (p < 0.05) and activity intensity (p < 0.05). When the physical activities were measured using Plus Cycle® in healthy cats, those data were quantified with high accuracy. In addition, it was also found to be very accurate in discriminating the resting and sleeping conditions of cats. In client-owned cats, there were no significant differences with respect to gender in any measured traits. The amount of physical activity and the number of jumps significantly decreased with the age of the cat. In contrast, the resting and sleeping times significantly increased with the age of the cat. In conclusion, Plus Cycle® can accurately and objectively assess physical activities and sleep quality with age of the cat, suggesting that this novel activity monitor can be used to manage the feline musculoskeletal health.