Yoshinori Nezu

Effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs.

OBJECTIVE To evaluate effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs. ANIMALS 12 healthy female Beagles. PROCEDURES A mid-diaphyseal transverse osteotomy (stabilized with an intramedullary pin) of the right tibia was performed in each dog. The carprofen group (n = 6 dogs) received carprofen (2.2 mg/kg, PO, q 12 h) for 120 days; the control group (6) received no treatment. Bone healing and change in callus area were assessed radiographically over time. Dogs were euthanized 120 days after surgery, and tibiae were evaluated biomechanically and histologically. RESULTS The osteotomy line was not evident in the control group on radiographs obtained 120 days after surgery. In contrast, the osteotomy line was still evident in the carprofen group. Callus area was significantly less in the carprofen group, compared with the area in the control group, at 20, 30, and 60 days after surgery. At 120 days after surgery, stiffness, elastic modulus, and flexural rigidity in the carprofen group were significantly lower than corresponding values in the control group. Furthermore, histologic evaluation revealed that the cartilage area within the callus in the carprofen group was significantly greater than that in the control group. CONCLUSIONS AND CLINICAL RELEVANCE Long-term administration of carprofen appeared to inhibit bone healing in dogs that underwent tibial osteotomy. We recommend caution for carprofen administration when treating fractures that have delays in healing associated with a reduction in osteogenesis as well as fractures associated with diseases that predispose animals to delays of osseous repair.

Trilostane-induced inhibition of cortisol secretion results in reduced negative feedback at the hypothalamic-pituitary axis.

Cushings disease caused by pituitary corticotroph adenoma in dogs is usually treated by medical treatment, and the efficacy of this treatment has been reported. However, controversy remains as to whether reduced negative feedback through the inhibition of cortisol secretion, similar to Nelsons syndrome, may appear as an adverse effect. The purpose of this study was to investigate the effect of reduced negative feedback through the inhibition of cortisol secretion by daily trilostane administration on the pituitary-adrenal axis in clinically normal dogs. Dogs were administered 5mg/kg trilostane twice a day every day for 8 weeks (n=8) or 16 weeks (n=3). After the initiation of trilostane administration, plasma adrenocorticotropic hormone (ACTH) concentrations were increased remarkably. As assessed by magnetic resonance imaging (MRI) during administration, the pituitary became enlarged. After trilostane administration, the cytoplasmic areas of the pituitary corticotrophs were increased and the ratio of pituitary corticotrophs to all cells in the anterior lobe was greater in the trilostane-treated dogs than that in untreated animals. In addition, histological examinations revealed bilateral adrenal cortical hyperplasia. Using real-time PCR quantification, the expression of proopiomelanocortin (POMC) mRNA in the pituitary and ACTH receptor (ACTH-R) mRNA in the adrenal gland was greater in the dogs treated with trilostane than in untreated dogs. These results indicate that reduced negative feedback induced hyperfunction of the pituitary corticotrophs and pituitary enlargement in healthy dogs. These changes suggest that the inhibition of cortisol secretion by trilostane may increase the risk for accelerating the growth of corticotroph adenomas in dogs with Cushings disease.

Effects of small intestinal ischemia and reperfusion on expression of tumor necrosis factor-α and interleukin-6 messenger RNAs in the jejunum, liver, and lungs of dogs

OBJECTIVE To determine the effects of intestinal ischemia and reperfusion on the expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNAs in the jejunum, liver, and lungs of dogs. ANIMALS 8 healthy adult Beagles. PROCEDURES In each dog, the cranial mesenteric artery was occluded for 0 (control group; n=4) or 60 (I-R group; 4) minutes, followed by reperfusion for 480 minutes; serum TNF-alpha and IL-6 activities and expression levels of TNF-alpha and IL-6 mRNAs in jejunal, hepatic, and lung tissues were measured before and at the end of the ischemic period and at intervals during reperfusion. For each variable, values were compared between the control and I-R groups at each time point. RESULTS Compared with the control group, serum IL-6 activity increased significantly after 180 minutes of reperfusion in the I-R group; also, jejunal TNF-alpha mRNA expression increased significantly after 60 (peak) and 180 minutes of reperfusion. In the I-R group, expressions of IL-6 mRNA in the liver and TNF-alpha and IL-6 mRNAs in the lungs increased significantly at 480 minutes of reperfusion, compared with the control group. Serum TNF-alpha activity, expression of IL-6 mRNA in the jejunum, and expression of TNF-alpha mRNA in the liver in the control and I-R groups did not differ. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the liver, lungs, and jejunum contributed to the production of TNF-alpha and IL-6 after intestinal ischemia and reperfusion in dogs, suggesting that intestinal ischemia and reperfusion induce a systemic proinflammatory cytokine response in dogs.

Conjunctival expression of the P2Y2 receptor and the effects of 3% diquafosol ophthalmic solution in dogs.

Conjunctival epithelial and goblet cell P2Y2 nucleotide receptors regulate ion transport and secretory function. Diquafosol is a P2Y2 purinergic receptor agonist that stimulates secretion of aqueous tear components from conjunctival epithelial cells and secretion of mucin from conjunctival goblet cells. In humans suffering from keratoconjunctivitis sicca (dry eye), topical administration of diquafosol improves corneal epithelial integrity and stabilises the tear film. The aim of the present study was to investigate P2Y2 receptor expression and to determine the effect of topical administration of diquafosol on mucin and aqueous tear production in dogs. Canine conjunctival P2Y2 receptor expression was evaluated by Western blotting and immunohistochemical analysis. The effect of diquafosol on mucin secretion was evaluated by examining mucin-5 subtype AC (MUC5AC) concentration in tears. The effect of diquafosol on aqueous secretions was evaluated by performing the Schirmer tear test (STT) and phenol red thread test. Expression of the P2Y2 receptor was confirmed in canine bulbar and palpebral conjunctivae and receptors were identified at the conjunctival epithelial and goblet cell surface. Tear MUC5AC concentration significantly increased after administration of 3% diquafosol ophthalmic solution, although neither STT nor phenol red thread test values showed any significant change after diquafosol instillation. Topical ocular administration of 3% diquafosol might improve corneal epithelial disorders in dogs through stabilisation of the tear film, by virtue of an increase in MUC5AC secretion.

Variations in Gene and Protein Expression in Canine Chondrodystrophic Nucleus Pulposus Cells following Long-Term Three-Dimensional Culture

Intervertebral disc (IVD) degeneration greatly affects quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP, because the cells disappear with age and are replaced by fibrochondrocyte-like cells. However, because IVD develops as early as within the first year of life, we used canines as a model to investigate in vitro the mechanisms underlying IVD degeneration. Specifically, we evaluated the potential of a three-dimensional (3D) culture of healthy NP as an in vitro model system to investigate the mechanisms of IVD degeneration. Agarose hydrogels were populated with healthy NP cells from beagles after performing magnetic resonance imaging, and mRNA expression profiles and pericellular extracellular matrix (ECM) protein distribution were determined. After 25 days of 3D culture, there was a tendency for redifferentiation into the native NP phenotype, and mRNA levels of Col2A1, COMP, and CK18 were not significantly different from those of freshly isolated cells. Our findings suggest that long-term 3D culture promoted chondrodystrophic NP redifferentiation through reconstruction of the pericellular microenvironment. Further, lipopolysaccharide (LPS) induced expression of TNF-α, MMP3, MMP13, VEGF, and PGES mRNA in the 3D cultures, creating a molecular milieu that mimics that of degenerated NP. These results suggest that this in vitro model represents a reliable and cost-effective tool for evaluating new therapies for disc degeneration.

Clinical efficacy of autogenous cancellous bone and fibroblast growth factor 2 combined with frozen allografts in femoral nonunion fractures

OBJECTIVES To evaluate the efficacy of cortical allograft and fibroblast growth factor 2 (FGF-2)-impregnated autogenous cancellous bone in nonunion fracture repair in dogs. METHODS From January 2000 to August 2010, seven dogs underwent cortical allograft and FGF-2-impregnated autogenous cancellous bone implantation for treatment of a femoral nonunion following fracture. Radiographic images were used to assess healing. RESULTS The average length of the implanted cortical allograft was 29.1 ± 4.4 mm. A significant improvement in the postoperative percentage of femoral shortening was observed with the experimental treatment, from 85.2 ± 8.2% to 95.0 ± 4.8%. Using radiographic scoring, we analysed the process of bone remodelling. At three months post-surgery, the proximal and distal fracture lines had begun to disappear, and a complete absence was observed after six months. Bacterial infection was detected in two of the seven cases. CLINICAL SIGNIFICANCE The findings of our study suggest that the combination of cortical allografts with FGF-2 impregnated cancellous autograft may be useful in cases of diaphyseal fracture non-union. The disappearance of the fracture line in dogs with nonunion was recognized at the same phase as the report in which healing process of allograft was evaluated in the experimental ostectomy model using the normal dog.

Biomarkers in dogs surgically treated for ruptured cranial cruciate ligaments

Thirty-one dogs with cranial cruciate ligament rupture (CCLR) were randomly treated with tibial plateau levelling osteotomy (TPLO; TPLO group) or proximal tibial osteotomy (PTO; PTO group). Synovial fluid was collected once before surgery and at one, two, three and six months after surgery. Cytokine activities (interleukin-1β, interleukin-6 and tumour necrosis factor-α) were determined by bioassays. Matrix metalloproteinase -3 activity was measured using a fluorogenic substrate. Sulphated glycosaminoglycan contents were determined by a dimethylmethylene blue dye-binding assay. Mediolateral and craniocaudal radiographs were obtained before surgery and at three and six months after surgery. Radiographic osteoarthritis findings were scored. Cytokine activities in the TPLO group appeared to decrease more quickly following surgery than those in the PTO group. At six months postoperatively, the progression of radiographic osteoarthritis score in the TPLO group was significantly lower than that in the PTO group. According to these results, joint inflammation in the stifle joints with CCLR could be reduced in the earlier postoperative period by performing TPLO, and TPLO could delay the progression of the radiographic findings of osteoarthritis, compared with PTO. TPLO may be an effective surgical procedure in the prevention of osteoarthritis progression in the stifle joints in dogs with CCLR. CCLR is one of the most common injuries in dogs and is the major cause of osteoarthritis in the stifle joint (Johnson and others 1994). The purposes of the surgical treatment of CCLR are to stabilise the stifle joint, to prevent secondary meniscal damage, and to inhibit the progression of osteoarthritis (Piermattei and others 2006). Intra- or extra-articular reconstructions of the cranial cruciate ligament (CCL) and corrective osteotomies of the tibia have been performed in dogs with CCLR. The main purpose of corrective osteotomies of the tibia is to provide functional stifle joint stability during the stance phase of the gait cycle …

In vitro evaluation of the relationship between the semitendinosus muscle and cranial cruciate ligament in canine cadavers

OBJECTIVE To evaluate the role of the semitendinosus muscle in stabilization of the canine stifle joint. SAMPLE Left stifle joints collected from cadavers of 8 healthy Beagles. PROCEDURES Left hind limbs, including the pelvis, were collected. To mimic the tensile force of the quadriceps, gastrocnemius, and semitendinosus muscles, wires were placed under strain between the ends of each muscle. A sensor was used to measure the tensile force in each wire. Specimens were tested in the following sequence: cranial cruciate ligament (CrCL) intact, CrCL transected, released (tensile force of semitendinosus muscle was released in the CrCL-transected stifle joint), and readjusted (tensile force of semitendinosus muscle was reapplied in the CrCL-transected stifle joint). Specimens were loaded at 65.3% of body weight, and tensile force in the wires as well as the cranial tibial displacement were measured. RESULTS Tensile force for the CrCL-transected condition increased significantly, compared with that for the CrCL-intact condition. Mean ± SD cranial tibial displacement for the CrCL-transected condition was 2.1 ± 1.3 mm, which increased to 7.2 ± 2.3 mm after release of the tensile force in the semitendinosus muscle. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the contention that the semitendinosus muscle is an agonist of the CrCL in the stifle joint of dogs. Moreover, the quadriceps and gastrocnemius muscles may be antagonists of the CrCL. These findings suggested that the risk of CrCL rupture may be increased by diseases (such as cauda equina syndrome) associated with a decrease in activity of the semitendinosus muscle.

Effect of rCaIFN-γ on NK cytotoxic activity in the peripheral blood of dogs

Previously, the ability of interferon (IFN) to reinforce antitumor immune capacity has received much attention. In humans and mice, natural killer (NK) cells are activated by IFN, thereby reinforcing antitumor immunity. We investigated whether NK cytotoxic activity can be enhanced by recombinant canine interferon-gamma (rCaIFN-γ) in dogs. First, we investigated the effects of various concentrations of and time exposures to IFN-γ in the culture medium on the NK cytotoxic activity of peripheral blood lymphocyte (PBLs) extracted from healthy beagles. Time- and concentration-dependent enhancement of NK cytotoxic activity of PBLs was observed. We then investigated whether the NK cytotoxic activity of PBLs is enhanced 24h after administration of rCaIFN-γ (10,000 units/kg body weight) in healthy beagles. Our in vivo study confirmed that NK cytotoxic activity of PBLs was enhanced by this approach, suggesting that antitumor immunity was reinforced. In dogs, rCaIFN-γ may be effective for bolstering antitumor immune capacity.

Efficacy of the use of a colorimetric pupil light reflex device in the diagnosis of fundus disease or optic pathway disease in dogs.

ABSTRACT The aim of this study was to determine the efficacy of a colorimetric pupil light reflex (PLR) device (Melan-100®, U.S.A.) in dogs with sudden acquired retinal degeneration syndrome (SARDS; 16 cases), progressive retinal atrophy (PRA; 10 cases) and optic pathway disease (6 cases). The colorimetric device detected PLR abnormality in 32, 16 and 9 eyes with SARDS, PRA and optic pathway disease, respectively, whereas white light detected PLR abnormality in 18, 11 and 9 eyes with SARDS, PRA and optic pathway disease, respectively. SARDS dogs displayed miosis, while optic pathway disease dogs displayed mydriasis in a blue light examination. Thus, colorimetric PLR may be a useful method for determining whether electroretinography (ERG) or magnetic resonance imaging (MRI) should be performed for dogs with acute blindness.