Yasushi Hara

Nippon Veterinary and Life Science University, Edo, Tōkyō, Japan

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Publications (95)100.57 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: OBJECTIVE To histologically evaluate and compare features of myofibers within the elongated soft palate (ESP) of brachycephalic and mesocephalic dogs with those in the soft palate of healthy dogs and to assess whether denervation or muscular dystrophy is associated with soft palate elongation. SAMPLE Soft palate specimens from 24 dogs with ESPs (obtained during surgical intervention) and from 14 healthy Beagles (control group). PROCEDURES All the soft palate specimens underwent histologic examination to assess myofiber atrophy, hypertrophy, hyalinization, and regeneration. The degrees of atrophy and hypertrophy were quantified on the basis of the coefficient of variation and the number of myofibers with hyalinization and regeneration. The specimens also underwent immunohistochemical analysis with anti-neurofilament or anti-dystrophin antibody to confirm the distribution of peripheral nerve branches innervating the palatine myofibers and myofiber dystrophin expression, respectively. RESULTS Myofiber atrophy, hypertrophy, hyalinization, and regeneration were identified in almost all the ESP specimens. Degrees of atrophy and hypertrophy were significantly greater in the ESP specimens, compared with the control specimens. There were fewer palatine peripheral nerve branches in the ESP specimens than in the control specimens. Almost all the myofibers in the ESP and control specimens were dystrophin positive. CONCLUSIONS AND CLINICAL RELEVANCE These results suggested that palatine myopathy in dogs may be caused, at least in part, by denervation of the palatine muscles and not by Duchenne- or Becker-type muscular dystrophy. These soft palate changes may contribute to upper airway collapse and the progression of brachycephalic airway obstructive syndrome.
    No preview · Article · Dec 2015 · American Journal of Veterinary Research
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    ABSTRACT: Objective-To identify characteristics of chondrodystrophoid and nonchondrodystrophoid small-breed dogs with cervical intervertebral disk herniation (IVDH). Design-Retrospective case series. Animals-187 small-breed (≤ 15 kg [33 lb]) dogs that underwent surgery because of cervical IVDH. Procedures-Medical records were reviewed for information on breed, sex, age, weight, location of affected intervertebral disks, duration and severity of neurologic signs, and recovery time. Results-55 of the 187 (29.4%) dogs were Beagles. The most frequently affected intervertebral disk was C2-3 (81/253 [32.0%]), and this was the more frequently affected intervertebral disk in dogs of several chondrodystrophoid breeds, including Beagles (29/66 [43.9%]), Dachshunds (13/37 [35.1%]), Shih Tzus (16/41 [39.0%]), and Pekingese (3/10 [30.0%]). However, caudal disks (C5-6 or C6-7) were more frequently affected in Yorkshire Terriers (13/24 [54.2%]) and Chihuahuas (9/13 [69%]). Shih Tzus and Yorkshire Terriers were significantly older at the time of surgery (mean ± SD age, 9.6 ± 2.3 years and 9.5 ± 2.5 years, respectively) than were Pomeranians (6.2 ± 2.3 years), and Yorkshire Terriers had a significantly higher number of affected disks (2.0 ± 0.9) than did Dachshunds (1.1 ± 0.3). Mean recovery time was significantly longer in Yorkshire Terriers (36.7 ± 13.0 days) than in Beagles (16.5 ± 17.1 days), Shih Tzus (17.8 ± 14.5 days), or Chihuahuas (12.2 ± 7. 2 days). Conclusions and Clinical Relevance-Results suggested that there may be breed-specific differences in the characteristics of cervical IVDH in small-breed dogs.
    No preview · Article · Dec 2015 · Journal of the American Veterinary Medical Association
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    ABSTRACT: Currently, the most commonly used bioresorbable scaffold is made of beta-tricalcium phosphate ( β -TCP); it is hoped that scaffolds made of a mixture of hydroxyapatite (HA) and poly-D/L-lactide (PDLLA) will be able to act as novel bioresorbable scaffolds. The aim of this study was to evaluate the utility of a HA/PDLLA scaffold compared to β -TCP, at a loading site. Dogs underwent surgery to replace a section of tibial bone with a bioresorbable scaffold. After the follow-up period, the scaffold was subjected to histological analysis. The HA/PDLLA scaffold showed similar bone formation and superior cell and tissue infiltration compared to the β -TCP scaffold, as seen after Villanueva Goldner staining. Moreover, silver staining and immunohistochemistry for Von Willebrand factor and cathepsin K demonstrated better cell infiltration in the HA/PDLLA scaffold. The fibrous tissue and cells that had infiltrated into the HA/PDLLA scaffold tested positive for collagen type I and RUNX2, respectively, indicating that the tissue and cells that had infiltrated into the HA/PDLLA scaffold had the potential to differentiate into bone. The HA/PDLLA scaffold is therefore likely to find clinical application as a new bioresorbable scaffold.
    Full-text · Article · Oct 2015
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    ABSTRACT: Osteoarthritis (OA) is a chronic degenerative joint disorder commonly associated with metabolic syndrome. As ageing and obesity has a great impact on the initiation/severity of OA, herein we sought to investigate the involvement of Sirt6 in the crosstalk between ageing and metabolic syndrome/OA. Sirt6 haploinsufficiency in mice promoted the expression of inflammatory cytokines in the IPFP. Enhanced inflammation of the IPFP in the aged Sirt6+/- HFD group was paralleled with accelerated OA change, including osteophyte growth and chondrocyte hypertrophy. Conversely, mesenchyme-specific Sirt6-deficient mice revealed both attenuated chondrocyte hypertrophy and proteoglycan synthesis, although chondrocyte senescence was enhanced as shown in the aged WT mice. Thus Sirt6 has key roles in the relationship among ageing, metabolic syndrome, and OA.
    Full-text · Article · Sep 2015 · Biochemical and Biophysical Research Communications
  • A. Sato · H. Ochi · Y. Harada · T. Yogo · N. Kanno · Y. Hara
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    ABSTRACT: The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease.
    No preview · Article · Sep 2015 · Domestic animal endocrinology
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    ABSTRACT: Objective: To determine whether thioredoxin (TRX)-1 can be used as a valid biomarker for oxidative stress in dogs. ANIMALS AND SAMPLES: 10 Beagles and Madin-Darby canine kidney cells. Procedures: Madin-Darby canine kidney cells were used to verify antigen cross-reactivity between human and canine anti-TRX-antibodies. Dogs were assigned to receive 21% or 100% O2 (5 dogs/group) via an artificial respirator during a 3-hour period of isoflurane anesthesia (starting at 0 hours). Blood and urine samples were collected before (baseline) and at 6, 12, 24, and 48 hours after commencement of inhalation anesthesia. Concentrations of TRX-1 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in plasma and urine samples were analyzed; urine concentrations were reported as ratios against urine creatinine concentration. Results: Canine TRX-1 was recognized by monoclonal human anti-TRX-1 antibodies (clones of adult T-cell leukemia-derived factor [ADF]-11 and ADF21) by western blot analysis. Results of an ELISA indicated that plasma TRX-1 concentration and urine TRX-1-to-creatinine concentration ratio increased rapidly after the 3-hour period of hyperoxia with maximal peaks at 12 and 6 hours, respectively. Urine 8-OHdG-to-creatinine concentration ratio also increased significantly after hyperoxia induction. However, unlike the rapid increase in urine TRX-1-to-creatinine concentration ratio, maximal urine 8-OHdG-to-creatinine concentration ratio was attained at 48 hours after hyperoxia induction. These variables remained unchanged from baseline in the control group. Conclusions and clinical relevance: Results indicated that human anti-TRX monoclonal antibodies cross-reacted with canine TRX, and plasma TRX-1 concentrations were rapidly increased in dogs following an oxidative stress challenge. Thus, TRX may be a valuable clinical biomarker for detecting oxidative stress more rapidly than 8-OHdG in dogs.
    Full-text · Article · Jun 2015 · American Journal of Veterinary Research
  • T Ichinohe · N Kanno · Y Harada · T Yogo · M Tagawa · Y Hara
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    ABSTRACT: To create a canine model of excessive tibial plateau angle (eTPA) and assess the chondroid metaplasia and extracellular matrix alteration in the cranial cruciate ligament. Seven mature female Beagles were included. Cylindrical osteotomy was performed bilaterally in the proximal tibia. The TPA was increased to approximately 40° in the left tibia (eTPA stifle) and left unchanged in the right tibia (control stifle). Exercise stress was started at three months postoperatively, and at 12 months postoperatively the dogs were euthanatized and the cranial cruciate ligaments were collected. The specimens were subjected to haematoxylin and eosin staining to assess the ligamentocyte morphology and immunostaining to assess the type I (COLI), type II (COLII), and type III (COLIII) collagen, and the sry-type HMG box 9 (SOX9) staining. Macroscopic cranial cruciate ligament injury was absent in six dogs but present in the eTPA stifle of one dog, which was excluded from the analysis. The ligamentocyte density decreased and the percentage of round ligamentocytes increased in the eTPA stifles. The COLII, COLIII, and SOX9 staining increased significantly and COLI deposition decreased in the eTPA stifles compared to the control stifle. The extracellular matrix changed, COLI deposition decreased, and COLIII and SOX9 staining increased in the cranial cruciate ligament of the eTPA stifles. SOX9 may contribute to COLII synthesis in the extracellular matrix of the cranial cruciate ligament in eTPA stifles, and eTPA may promote chondroid metaplasia and extracellular matrix alteration.
    No preview · Article · May 2015 · Veterinary and Comparative Orthopaedics and Traumatology
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    ABSTRACT: Rehabilitation is drawing more and more attention as a therapeutic practice, also in veterinary surgery. In this study, we performed post-operative rehabilitation on a dog which had gone through tibial-plateau-leveling osteotomy (TPLO), and whose treatment had been complicated by cranial cruciate ligament rupture and cauda equina syndrome. We then examined the usefulness of rehabilitation. We performed icing, passive range-of-motion (PROM) exercise, massotherapy, hyperthermia and hydropathy on the dog from the postoperative early stage. The results revealed that the range of joint motion and the muscle mass decreased less than in the control dogs. Time to recovery of walking, analyzed with the force plate, was two months, which was shorter than in the control dogs, which was three months. Collectively, we suggest that the rehabilitation at the early stage is effective in accelerating postoperative recovery. We hope that rehabilitation will become more common in veterinary medicine also in Japan, and it will be helpful for patient animals to become free of paralysis and/or pain, and to reduce the suffering of owners.
    No preview · Article · Mar 2015
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    ABSTRACT: 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.
    Full-text · Article · Feb 2015 · Journal of Veterinary Medical Science
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    ABSTRACT: Intervertebral disc degeneration (IVDD) greatly affects the 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. Because IVDD develops as early as within the first year of life, we used canines as a model to investigate the in vitro mechanisms underlying IVDD. The mechanism underlying age-related IVDD, however, is poorly understood. Several research groups have suggested that Wnt/β-catenin signaling plays an important role in IVDD. However, the role of Wnt/β-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/β-catenin signaling could enhance Runx2 expression in IVDD and lead to IVD calcification. NP tissue was obtained from Beagle dogs after evaluation of the degeneration based on magnetic resonance imaging (MRI). Histological analysis showed that lack of Safranin-O staining, calcified area, and matrix metalloproteinase13-positive cells increased with progression of the degeneration. Furthermore, the levels of β-catenin- and Runx2-positive cells also increased. Real-time reverse-transcription polymerase chain reaction analysis showed that the MRI signal intensity and mRNA expression levels of β-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced β-catenin accumulation and Runx2 expression. In contrast, FH535 inhibited LiCl-induced upregulation. These results suggest that Runx2 transcript and protein expression, potentially in combination with β-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs. © 2014 Wiley Periodicals, Inc.
    No preview · Article · Jan 2015 · Journal of Cellular Physiology
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    ABSTRACT: 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 stabilizes 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 found 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 though stabilization of the tear film, by virtue of an increase in MUC5AC secretion.
    No preview · Article · Oct 2014 · The Veterinary Journal
  • N Kanno · Y Hara · S Fukano · H Fujie · H Ochi · Y Fujita · H Yasuji · Y Nezu · T Yogo · M Tagawa
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    ABSTRACT: Objective: The aim of this study was to investigate the biomechanical effects of cranial cruciate ligament (CrCL) transection on stifle stability at three different stifle joint flexion angles using a robotic system. Methods: This was an ex vivo biomechanical study. Stifles (n = 6) were collected from the cadavers of Beagles weighing 10.5-12.0 kg. Six stifle joints were dissected, potted, and secured to the manipulator arms of a robotic simulator. With the stifle joint angle maintained at either hyperextension (151°), 135° or 90°, stability was assessed by application of a 50 N load in either the cranial-caudal (CrCd test) or proximal-distal (PD test) directions. The stifle was given a cranial-caudal load of 50 N (CrCd test). A proximal-distal compression load of 50 N was then administered by the manipulator (proximal-distal test: PD test). The change in three-dimensional kinematics of the intact and the CrCL-transected stifles was compared between hyperextension, and 135° and 90° flexion for the CrCd and PD load conditions. A value of p <0.05 was considered statistically significant. Results: The cranial tibial displacements in the PD tests of the CrCL-transected stifles at 135° (8.4 ± 1.2 mm) and at 90° (8.1 ± 1.9 mm) were significantly greater than the displacement at 151.5° (5.1 ± 1.6 mm) (p = 0.004 and p = 0.012 respectively). Clinical significance: The canine stifle exhibited the most instability when the stifle flexion angle was 135°.
    No preview · Article · Jul 2014 · Veterinary and Comparative Orthopaedics and Traumatology
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    ABSTRACT: Objectives: In dogs with deep analgesia caused by acute spinal cord injury from thoracolumbar disk herniation, autologous bone marrow mononuclear cell transplant may improve recovery. The purpose of the present study was to evaluate autologous bone marrow mononuclear cell transplant in a dog that had paraplegia and deep analgesia caused by chronic spinal cord injury. Materials and methods: Autologous bone marrow mononuclear cell transplant was performed in a dog having paraplegia and analgesia for 3 years that was caused by a chronic spinal cord injury secondary to Hansen type I thoracolumbar disk herniation. Functional recovery was evaluated with electrophysiologic studies and the Texas Spinal Cord Injury Scale. Results: Somatosensory evoked potentials were absent before transplant but were detected after transplant. Functional improvement was noted (Texas Spinal Cord Injury Scale: before transplant, 0; after transplant, 6). No adverse events were observed. Conclusions: Autologous bone marrow mononuclear cell transplant into the subarachnoid space may be a safe and beneficial treatment for chronic spinal cord injury in dogs.
    No preview · Article · Jun 2014 · Experimental and clinical transplantation: official journal of the Middle East Society for Organ Transplantation
  • Munetaka Iwata · Takaharu Hakozaki · Yasushi Hara
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    ABSTRACT: A four-year-old male toy poodle presented with signs of cervical pain over a period of two years, with subsequent progression to tetraparesis. Neurological examination suggested a transverse myelopathy in the cervical spinal cord. Radiographs of the cervical spine showed fractured endplates of the third through the sixth cervical vertebrae and delayed growth plate closure of all skeletal structures. Total serum thyroid-stimulating hormone and T4 concentrations were very low, indicating hypothyroidism. MRI revealed a pituitary gland cyst and spinal cord compression by fractured metaphysis which were displaced dorsally into the spinal canal. Surgical treatment resulted in marked improvement in clinical signs. Open physes were nearly closed 16 months after continuous administration of levothyroxine sodium hydrate. Based on these findings, a presumed diagnosis of Salter–Harris type III fractures of the vertebral endplates associated with delayed ossification of the growth plates due to hypothyroidism was made.
    No preview · Article · Feb 2014
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    ABSTRACT: To compare data for French Bulldogs and Dachshunds that had hemilaminectomy for thoracolumbar intervertebral disc extrusion (T-L IVDE) by 1 surgeon and to evaluate the association between IVDE and congenital vertebral anomalies. Retrospective case series. French Bulldogs (n = 47) and 671 Dachshunds. Age, gender, vertebral anomaly, kyphosis/kyphoscoliosis, IVDE site, non-recovery and progressive hemorrhagic myelomalacia development from grade 5 (paraplegia without deep nociception) were compared between the 2 breeds. French Bulldogs were significantly younger (P = .00001), more likely to be male (P = .023), and more likely to have a congenital vertebral anomaly and kyphosis/kyphoscoliosis (P < .00001) than Dachshunds. The frequencies of French Bulldogs with IVDE within typical sites (T11-L3) were significantly lower (P = .0005) and within caudal sites (L3-L7) significantly higher (P = .0001) compared with Dachshunds. None of the French Bulldogs had IVDE within the kyphotic/kyphoscoliotic segment. The frequency of lumbar IVDE (L1-L5) in French Bulldogs with kyphosis/kyphoscoliosis was significantly higher (P = .003) compared with French Bulldogs without kyphosis/kyphoscoliosis. In grade 5 dogs, the risk of developing progressive hemorrhagic myelomalacia in French Bulldogs was significantly higher (P = .03) than in Dachshunds. The distribution of IVDE site in French Bulldogs within the thoracolumbar and lumbar spine was different from Dachshunds. IVDE sites were not located at the sites of vertebral anomaly. French Bulldogs appeared to have T-L IVDE at younger ages, with higher male predisposition and higher risk of developing progressive hemorrhagic myelomalacia from grade 5 compared with Dachshunds.
    No preview · Article · Jan 2014 · Veterinary Surgery
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    ABSTRACT: 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 (10,000units/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.
    No preview · Article · Sep 2013 · Research in Veterinary Science
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    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.
    Preview · Article · Jun 2013 · Journal of Veterinary Medical Science
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    ABSTRACT: Hydroxyapatite (HA)/poly-l-lactide(PLLA) composite biomaterials are available for orthopedic applications, but bioresorption and cell-mediated inflammation in bone cortex are unknown. We conducted an 84-month follow-up study with Beagle dogs that were subjected to implants with either PLLA (left femur) or HA/PLLA (right femur). Histological and radiographic analysis showed that HA/PLLA screws induced significant increases in HA content from 36 months onward and complete burr hole closure at 60 months, whereas PLLA screws did not. Moreover, PLLA screws induced more severe fibrous tissue and histiocyte infiltration. HA/PLLA screws promote earlier burr hole replacement and have superior biocompatibility compared to PLLA screws.
    No preview · Article · May 2013 · Journal of Biomaterials Applications
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    ABSTRACT: 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.
    Full-text · Article · May 2013 · PLoS ONE
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    ABSTRACT: Obesity and high body mass index are associated with a higher incidence of osteoarthritis (OA). The aim of this study is to investigate the involvement of the infrapatellar fat pad (IPFP) in the sub-acute effect of a high fat diet (HFD) on the development of knee-OA. C57BL/6J male mice were fed either a HFD or a normal diet beginning at seven weeks of age. Tissue sections were evaluated with immunohistological analysis. The IPFP was excised, and mRNA expression profiles were compared using real-time RT-PCR analysis. Osteoarthritic changes were initiated in the HFD group after eight weeks of the HFD. Increased synovial cell number and angiogenesis at the anterior edge of the tibial plateau were exhibited prior to osteophyte formation. Quantitative histological analysis indicated that osteophyte volume was significantly increased in the HFD group after eight weeks, along with an increase in the IPFP volume, the size of individual adipocytes and the number of vessels in the IPFP. Histomorphometrical analysis revealed osteophyte area was significantly associated with IPFP area, individual adipocyte area and vascular area. Real-time RT-PCR analysis demonstrated elevated mRNA expression of inflammatory cytokines, growth factor, and adipokines in the IPFP after eight weeks of the HFD. These findings are in parallel with increased expression of the CD68 macrophage marker after eight weeks of the HFD. Expression levels of the adipokines were significantly correlated with expression of TNF-α, VEGF and TGF-β. Immunohistological analysis revealed that the Nampt protein was highly expressed in the IPFP especially around the site of osteophyte formation. Apoptosis and proliferation of chondrocytes were both enhanced at the site of osteophyte formation, indicating higher cell turnover at this region. These observations suggest the IPFP plays a pivotal role in the formation of osteophytes and functions as a secretory organ in response to a HFD.
    Full-text · Article · Apr 2013 · PLoS ONE