Yasushi Hara

The aim of this study was to determine the efficacy of a colorimetric pupil light reflex (PLR) device (Melan-100 (R), 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.

To investigate influence of general anesthesia on immunological anti-tumor activity, the natural killer (NK) cytotoxic activity of peripheral lymphocytes (PBLs) was measured in 7 dogs anesthetized for 3 hr with isoflurane following propofol-induction (anesthesia group) and 6 dogs without anesthesia (control group). Blood samples were collected before (baseline) and 24, 120 and 192 hr after the anesthesia. The PBLs were isolated via centrifugation with Ficoll-Hypaque solution (density, 1.073), and adherent cells were removed. The NK cytotoxic activity of the isolated PBLs against canine thyroid cancer cells was detected by the colorimetric rose Bengal assay. Significant decrease in the NK cytotoxic activity was observed at 24 hr after the anesthesia, compared with the baseline values and the control group. The NK cytotoxic activities were recovered to the baseline values until 120 hr after the anesthesia. The general anesthesia with isoflurane following propofol-induction decreased the NK cytotoxic activities of PBLs in dogs. This finding has a clinical relevance to the risk of tumor recurrence or metastasis induced by the suppression of immunological anti-tumor activity after general anesthesia in dogs. The results further emphasized the importance of the need to evaluate immune suppression following general anesthesia in animals.

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-alpha, 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.

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-alpha, VEGF and TGF-beta. 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.

Akagi H, Ochi H, Kannno N, Iwata M, Ichinohe T, Harada Y, Nezu Y, Yogo T, Tagawa M, Hara Y, Veterinary and comparative orthopaedics and traumatology : V.C.O.T, 2013, vol. 26, no. 2, pp. 123-129

Objective-To investigate the relationship between runt-related transcription factor 2 (RUNX2) expression in canine nucleus pulposus (NP) cells and intervertebral disk aging in chondrodystrophoid dogs. Animals-7 healthy Beagles (mean age, 35.6 months) and 11 Dachshunds with herniated disks (mean age, 61 months). Procedures-All dogs underwent MRI examination of the thoracic and lumbar vertebral column immediately before sample collection under general anesthesia. The disk center-to-CSF T2-weighted signal intensity ratio was determined for healthy Beagles. Samples of NP were obtained from nonherniated disks in healthy Beagles and from herniated disks during surgical treatment of hospitalized Dachshunds. Samples were evaluated for RUNX2 and matrix metalloproteinase 13 transcript expression via reverse transcriptase PCR assay; RUNX2 protein expression was evaluated via immunohistochemical analysis, and correlation between these variables and age of dogs was evaluated. A 3' and 5' rapid amplification of cDNA ends method was used to identify the RUNX2 coding region. Results-RUNX2 cDNA had > 97% conservation with the human cDNA sequence and approximately 95% conservation with the mouse cDNA sequence; RUNX2 and matrix metalloproteinase 13 mRNA expression and RUNX2 protein expression in NP cells were positively correlated with age. The disk center-to-CSF T2-weighted signal intensity ratio was negatively correlated with RUNX2 protein expression in the NP of healthy dogs. Conclusions and Clinical Relevance-Results indicated that RUNX2 mRNA and protein expression in the NP are enhanced in aging intervertebral disks in dogs. (Am J Vet Res 2012;73:1553-1559)

Because bone morphogenetic protein 2 gene transfected Escherichia coli (E-BMP-2) produce recombinant human BMP-2 (rhBMP-2) more efficiently than mammalian cells (Chinese hamster ovary [CHO]-BMP-2), they may be a more cost-effective source of rhBMP-2 for clinical use. However, use of E-BMP-2 for regenerating long bones in large animals has not been reported. In the current study, we evaluated the healing efficacy of E-BMP-2 in a canine model. We created 2.5-cm critical-size segmental ulnar defects in test animals, then implanted E-BMP-2 and 700 mg of artificial bone (beta-tricalcium phosphate; beta-TCP) into the wounds. We examined the differential effects of 5 E-BMP-2 treatments (0, 35, 140, 560, and 2240 mu g) across 5 experimental groups (control, BMP35, BMP140, BMP560, and BMP2240). Radiography and computed tomography were used to observe the regeneration process. The groups in which higher doses of E-BMP-2 were administered (BMP560 and BMP2240) displayed more pronounced bone regeneration; the regenerated tissues connected to the host bone, and the cross-sectional areas of the regenerated bone were larger than those of the originals. The groups in which lower doses of E-BMP-2 were administered (BMP35 and BMP140) experienced relatively less bone regeneration; furthermore, the regenerated tissues failed to connect to the host bone. In these groups, the cross-sectional areas of the regenerated bone were equal to or smaller than those of the originals. No regeneration was observed in the control group. These findings suggest that, like CHO-BMP-2, E-BMP-2 can be used for the regeneration of large defects in long bones and that its clinical use might decrease the cost of bone regeneration treatments.

Objectives: Severe intervertebral disc herniation causes complete paraplegia and loss of pain sensation in canines. The prognosis is poor, even when decompression surgery is performed immediately after onset. Studies suggest that bone marrow-derived mononuclear cells will regenerate the injured spinal cord and restore neurologic function. This study was conducted to assess the clinical efficacy of bone marrow-derived mononuclear cell autotransplanting in severe cases of canine intervertebral disc herniation. Materials and Methods: Eighty-two dogs (miniature dachshunds) with severe thoracolumbar intervertebral disc herniation were used. All had intervertebral disc herniation accompanied by paraplegia and loss of pain perception. In 36 dogs, bone marrow-derived mononuclear cells were autotransplanted to the lesioned spinal cord immediately after decompression surgery. Bone marrow was collected from the proximal humerus and subjected to density gradient centrifugation to isolate the bone marrow-derived mononuclear cells. The remaining 46 dogs (receiving surgical treatment only) were assigned as controls. Therapeutic efficacy was compared based on the rate of ambulatory recovery. Results: Ambulatory recovery was observed in 88.9% and 56.5% of animals in the bone marrow-derived mononuclear cells and control groups, and a significant difference was found. No complications were found in bone marrow-derived mononuclear cells group. Conclusions: Bone marrow-derived mononuclear cell transplanting revealed a significant increase in the recovery rate and, as has been reported in rats and humans, bone marrow-derived mononuclear cell autotransplanting shows efficacy in canines as well.

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 72 +/- 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. (Am J Vet Res 2012;73:672-680)

This study was undertaken to investigate the age-related differences of monocyte chemotactic protein-1 (MCP-1) and matrix metalloproteinase-3 (MMP-3) expression in mouse intervertebral disc (IVD) and to determine whether MMP-3 plays a role in disc degeneration. Expression of MCP-1 and MMP-3 mRNA in mouse IVD was assessed by quantitative PCR. The ability of MCP-1 and MMP-3 expression in IVD to respond to TNF-α or TWEAK stimulation was examined by quantitative PCR, WB, ELISA, and immunohistochemistry. IVD derived from MMP-3-deficient and wild-type mice were compared using Safranin-O staining and immunohistochemistry. mRNA levels of MCP-1 and MMP-3 in IVD significantly diminished and the ability of MCP-1 or MMP-3 expression to respond to TNF-α or TWEAK stimulation was significantly reduced as age increased. IVD derived from 64-week-old wild-type mice showed clearly diffuse proteoglycan loss by Safranin-O staining and immunohistochemistry compared with younger mice. However, no loss of proteoglycan and typeII collagen were observed in IVD derived from 64-week-old MMP-3-deficient mice. MCP-1 and MMP-3 expression in mouse IVD showed age-related decreases. The response to inflammation in IVD also displayed age-related changes. Therefore, disc degeneration may vary with the patients' age and targeting MMP-3 may be a possible future therapeutic strategy for disc degeneration. Copyright © 2011 Orthopaedic Research Society.

The displacement and signal intensity (SI) of the pituitary posterior lobe were evaluated on TI weighted magnetic resonance (MR) images in 28 dogs with pituitary dependent hyperadrenocorticism (PDH). Compared with normal dogs, the posterior lobe was displaced dorsally in the pituitary of the PDH dogs. Correlation between the pituitary height/brain area (P/B) ratio and the displacement of the posterior lobe in the PDH dogs suggests that dorsal displacement of the posterior lobe increases in accordance with enlargement of the pituitary. As to the SI of the posterior lobe, the PDH dogs showed significantly lower SI in comparison to the normal dogs. Taken together, these results suggest that assessment of the displacement and SI of the posterior lobe of the pituitary on TI-weighted MR images is useful for the diagnosis of pituitary adenoma. In pituitary microadenoma that presents no apparent tumorigenesis on M RI, evaluation of these values may be useful for diagnosis and selection of an appropriate therapy.

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. (Am J Vet Res 2011;72:634-641)

This study investigated the influence of dried okara-tempeh, the insoluble portion of soybeans fermented with ragi-tempeh, on fecal microbiota and metabolites in dogs. Three dried okara-tempehs (individual weight: approximately 5 g) were given daily to each of 6 beagle dogs for a period of 2 weeks. The concentrations of fecal short-chain fatty acids, pH, and microbiota were determined. During the intake of the dried okara-tempeh, the level of Bifidobacterium expressed as log(10) number (CFU)/g wet feces was increased significantly (p<0.05) from 6.4 +/- 0.9 (mean +/- SD) to 8.8 +/- 0.8, and the level of Bacillus was also increased significantly (p < 0.05) from 4.1 +/- 1.8 to 7.3 +/- 0.6. Moreover, fecal concentrations of total short-chain fatty acids, acetic acid and propionic acid also increased significantly (p < 0.05) from 146.7 +/- 15.9 mu mol/g wet feces to 198.8 +/- 24.6 mu mol/g wet feces, from 90.3 +/- 8.6 mu mol/g wet feces to 116.3 +/- 17.91 mu mol/g wet feces, and from 39.3 +/- 3.7 mu mol/g wet feces to 60.0 +/- 9.1 mu mol/g wet feces respectively on day 7 of dried okara-tempeh intake compared to pre-intake. Fecal pH decreased significantly (p < 0.05) from 6.2 +/- 0.3 to 5.6 +/- 0.2 during intake. The findings obtained by this study demonstrate that the intake of dried okara-tempeh was effective for improving the fecal environment in dogs and that okara, which is currently discarded, can be effectively utilized in dog feed.

Transsphenoidal surgery (TSS) is an effective treatment for canine Cushing's disease, as well as human Cushing's disease. In humans, only the pituitary adenoma tissue is resected by TSS. However, in dogs, the whole pituitary including normal tissue is resected. Hence, central diabetes insipidus (CDI) may complicate the postoperative course in almost all dogs with Cushing's disease treated by TSS. However, it is difficult to assess the duration of the postoperative CM, and whether it may be transient or permanent. In this study, postoperative CDI in 21 dogs with Cushing's disease and its predicted prognosis by preoperative parameters was investigated. In this study, CDI after TSS was classified as either transient or permanent based on the requirement for desmopressin. Preoperative circulating serum cortisol concentrations and pre- and post-operative plasma AVP concentrations were not significantly different between the transient CDI dogs and permanent CDI dogs. The duration of postoperative CD! was not correlated to the signal intensity ratio (posterior lobe of the pituitary/cerebral cortex), which is obtained from preoperative magnetic resonance imaging (MRI). However, the pituitary height/brain area (P/B) ratio of the permanent CDI dogs was significantly greater than that of the transient CDI dogs. In addition, there was a significant difference between the enlarged-pituitary (P/B ratio > 0.31) transient CDI dogs and the permanent CDI dogs. These results suggest that the incidence of postoperative permanent CDI is strongly influenced by the pituitary size.

LPS (lipopolysaccharide), a major constituent of Gram-negative bacteria, regulates proliferation and differentiation of osteoclasts directly or indirectly. This study sought to investigate the functions of the RANK/RANKL pathway in LPS-induced bone loss in vivo. Wild-type mice or TNFR1-/- mice were injected LPS with or without osteoprotegerin (OPG) and analyzed histologically. Bone volume was reduced by LPS injection in all groups, and OPG administration prevented the LPS-induced bone loss regardless of genotypes. LPS-induced enhancement of osteoclastogenesis in wild-type mice was blocked by OPG administration. LPS or OPG did not affect osteoclastogenesis in TNFR1-/- mice. Interestingly, osteoblast surface was remarkably reduced in LPS-treated TNFR1-/- mice as a result of enhanced osteoblast apoptosis. TRAIL, induced by TNF-alpha. in BMC, triggered apoptosis of primary osteoblast only when TNFR1 signal was ablated in vitro. In conclusion, RANK signaling plays a prominent role in osteoclastogenesis downstream of LPS. Furthermore, TNFR1 regulates bone metabolism through not only the regulation of osteoclast differentiation but also osteoblast survival. (C) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:657-663, 2010

Postoperative changes in endocrinological status and serum chemistry during the 4 years after transsphenoidal surgery (ISS) in 25 dogs with Cushing's disease were investigated in a prospective study. In all 25 dogs. Cushing's disease was diagnosed from resected pituitary tissues as a corticotroph adenoma in the anterior lobe of the pituitary. Prior to TSS, all 25 clogs showed hypercortisolemia. After TSS, the ACTH stimulation test showed continued low serum cortisol concentrations in 21 dogs (84%). In addition, the serum thyroid stimulating hormone concentrations decreased sequentially, while the serum T4 concentrations tended to increase due to the postoperative hormone substitution therapy utilized to avoid secondary hypothyroidism. In recital to serum chemistry. alkaline phosphatase (ALP), alanine aminotransferase (ALT) and total cholesterol are commonly increased in can Cushing's disease. In this study. ALP. ALT and total cholesterol were increased in 23 eases (92%), 19 cases (76%) and 20 cases (80%). respectively. However. postoperatively, these concentrations gradually decreased. The postoperative serum concentrations of ALP at I year, that of ALT at six months to 2 years and that of total cholesterol over the course of the 4 years decreased significantly compared with the concentrations before TSS. These results show that TSS is an effective treatment for canine Cushing's disease and for long-term improvement of hypercortisolemia. Moreover. TSS is effective in improvement or hypercortisolism, such as increased concentrations of serum ALP. ALT and total cholesterol.

An 11-year-old male mixed breed dog diagnosed with Gushing's disease and diabetes mellitus was treated by hypophysectomy After surgery. the hypercortisolemia disappeared and the diabetes status improved The insulin requirement to control hyperglycemia gradually decreased At 12 weeks after surgery, there was no requirement for insulin and we suspected the diabetes was completely resolved In the present case diabetes mellitus seems to be secondary to Cushing's disease In conclusion, this mixed breed dog with coexisting Cushing's disease and diabetes mellitus is the first case showing the effectiveness of hypophysectomy to treat diabetes mellitus secondary to Cushing's disease in dogs

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in porcine iris sphincter were investigated. Forskolin and sodium nitroprusside inhibited carbachol (CCh)-induced contraction in a concentration-dependent manner. Various selective PDE inhibitors, vinpocetine (type 1), erythro -9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4) and zaprinast (type 5), also inhibited CCh-induced contraction in a concentration-dependent manner. The rank order of potency of IC(50) was zaprinast > Ro20-1724 > EHNA >= milrinone > vinpocetine. In the presence of CCh (0.3 mu M), vinpocetine, milrinone and Ro20-1724 increased cAMP, but not cGMP, contents. In contrast, zaprinast and EHNA both increased cGMP, but not cAMP, contents. This indicates that vinpocetine-, milrinone- and Ro20-1724-induced relaxation is correlated With cAMP, While EHNA- and zaprinast-induced relaxation is correlated with cGMP in porcine iris sphincter.

Fourteen hip joints of 8 juvenile dogs with hip dysplasia were evaluated by radiographic and arthroscopic examinations. The purpose of the study was to evaluate the relationship between radiographic and arthroscopic findings. Radiographic hip osteoarthritis grades were obtained from the ventrodorsal hip-extended view. The distraction index (DI) for each hip joint was calculated from the compression and distraction views. Articular cartilage pathology, the condition of the ligament of the femoral head and the synovium were evaluated by arthroscopy. Results showed that dogs with higher DIs were likely to have more severe cartilage injuries in the hip joints. Hip arthroscopy was useful for evaluating joint pathology and the findings seem to correlate with joint laxity in young dogs with hip dysplasia.<br>

Cushing&apos;s disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing&apos;s disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells. ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC). corticotropin releasing hormone receptor 1 (CRHR 1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT PCR. POMC, CRHR 1, and 11 HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold: 11HSD2. 4.2-fold, P&lt;0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR. 2.2-fold: 11HSD1, 2.9-fold, P&lt;0.01, respectively). GR mRNA levels did not (filter between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing&apos;s disease. (C) 2008 Elsevier Inc. All rights reserved.

Cushing&apos;s 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 Nelson&apos;s 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 5 mg/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 Cushing&apos;s disease. (C) 2008 Elsevier Inc. All rights reserved.

Pituitary thyrotroph hyperplasia results from prolonged primary hypothyroidism in humans, mice and rats. In dogs with Cushing&apos;s disease, many cases have low serum thyroid hormones concentrations due to euthyroid sick syndrome. A 6-year-old castrated male Beagle diagnosed with Cushing&apos;s disease had a high serum thyroid Stimulating hormone (TSH) concentration that was treated by hypophysectomy. On histological examination, the resected pituitary gland contained both a corticotroph adenoma and thyrotroph hyperplasia. The TSH-positive cell ratio in this case was greater than that of healthy Beagles. In the present case, the pituitary thyrotroph hyperplasia was probably caused by primary hypothyroidism. In conclusion, this Beagle is the first histological confirmation of the coexistence of a corticotroph adenoma and thyrotroph hyperplasia.

Cerebrospinal fluid pressure (CSFP) in the cervix and lumbar spine was measured percutaneously using a fiberoptic pressure monitor in 28 healthy dogs without neurological abnormalities. The cervical CSFP (C-CSFP) ranged from 2.0 to 12.0 mmHg and its mean was 6.11 mmHg, while the lumber CSFP (L-CSFP) ranged from 2.0 mmHg to 12.0 mmHg and its mean was 5.93 mmHg. The ranges of C-CSFP and L-CSFP were similar. The mean arterial blood pressure (BP) increased from 127.5 ± 14.1 mmHg to 203.9 ± 33.3 mmHg immediately after epinephrine administration, then returned to 122.7 ± 25 mmHg at 30 min after epinephrine administration. The mean C-CSFP was 8.4 ± 3.4 before epinephrine administration, 10.0 ± 5.1 and 9.4 ± 4.8 mmHg at 0 and 30 min after epinephrine administration, respectively. However, the mean L-CSFP was 4.4 ± 2.8 before epinephrine administration, 4.6 ± 5.7 and 5.6 ± 5.0 mmHg at 0 and 30 min after epinephrine administration, respectively. There was no correlation among C-CSFP, L-CSFP, and arterial BP. This study demonstrated that the percutaneous fiberoptic catheter method allows stable and continuous monitoring of CSFP in the spinal canal in dogs.<br>

The posterior lobe of the pituitary gland (PL) normally shows characteristic high signal intensity (SI) on T1-weighted MR images (T1WI) in humans. The high S1 is thought to represent storage of arginine vasopressin (AVP) in the PL. Normal dogs also show a high SI on T1WIs, but the origin is unclear. In the present study, we investigated whether the high Sl in the PL on T1WIs in normal dogs is caused by AVP. We examined the Sl in the PL on T1WIs. plasma AVP concentrations and plasma osmolality in normal dogs after excessive AVP secretion was induced by hypertonic saline overload. In addition, functional changes in the supraoptic nucleus and paraventricular nucleus of the hypothalamus under AVP secretion-stimulated conditions were examined immunohistologically. Under hypertonic saline overload, plasma osmolality and plasma AVP concentrations gradually increased, while the Sl of the PL gradually decreased. This suggests that AVP secretion was stimulated by elevated osmolality. Moreover, there was a significant negative correlation between plasma AVP concentrations and the SI ratio of the PL. An immunohistochemical study of the hypothalamus nucleus revealed that AVP-immunopositive cells significantly increased in the hypertonic saline loaded dogs. We concluded that the high SI in the PL in TIWIs in normal dogs was caused by AVP stored at the site, and examination of the Sl in the PL using MRI is useful for diagnosis of abnormal pituitary glands.

We investigated the epidemiological characteristics of intervertebral disc herniation (IVDH) in Japan in a large population using a retrospective study. The sample population was dogs (n=297) with IVDH in Japan. Medical records were reviewed for breed, sex, age, affected interspace and neurological severity. The dogs were comprised of 132 cases of cervical IVDH (C-IVDH) and 165 cases of thoracolumbar IVDH (TL-IVDH). In Japan, the Dachshund, Beagle and Shih Tzu tended to suffer from both C-IVDH and TL-IVDH. The Shiba Inu, a characteristic Japanese dog breed, suffered from both C-IVDH and TL-IVDH, although there was little data relating to the whole breed. Male dogs tended to suffer from C-IVDH and TL-IVDH at a rate almost twice that of females in Japan. Among the three predominant dog breeds, the Dachshund, Beagle and Shih Tzu, the Dachshund tended to suffer from both C-IVDH and TL-IVDH at an earlier age than the Beagle, and the Beagle tended to Suffer from both C-IVDH and TL-IVDH earlier than the Shih Tzu. Among the three predominant breeds, the Shih Tzu, in particular, tended to suffer from both C-IVDH and TL-IVDH at multiple sites. Our data from Japan were in partial agreement with previous data from the U.S.A., and epidemiological characteristics of IVDH peculiar to Japan were also identified.

Thrombosis is a potential complication of hyperadrenocorticism (HAC) in dogs. An 8-year-old male Beagle diagnosed with pituitary-dependent HAC had complicated with thrombosis in the caudal vena cava and abdominal aorta, which was treated by hypophysectomy and antithrombotic therapy. After hypophysectomy, hypercortisolemia disappeared and the general condition was also significantly improved. Ultrasonography after hypophysectomy revealed that the thrombus remained in the abdominal aorta, but the thrombus in the caudal vena cava had disappeared. However 692 days after the hypophysectomy, the dog had an acute onset of dyspnea and died. Postmortem examination revealed the presence of thrombi in the abdominal aorta and the pulmonary artery. Observations from this case show that HAC dogs must be attention to thrombosis.

The causes of early degeneration and calcification of the nucleus pulposus in the chondrodystrophoid dog are poorly understood, and the underlying molecular mechanism of this process has not yet been clearly defined. Type X collagen is one of the key molecules in endochondral bone growth and development, especially matrix calcification. The relationship between type X collagen and disc degeneration and calcification in chondrodystrophoid dogs has not yet been studied. We analyzed the expression of type X collagen in degeneration and calcification of the intervertebral disc in chondrodystrophoid dogs, using type X collagen immunohistochemistry. Control intervertebral discs were collected from five dogs (4 female, 1 male, average age 1.3 years, beagle breed). Degenerated intervertebral discs were urgically removed from 11 canine patients with intervertebral disc extrusion (1 female, 10 male, average age 5.1 years, dachshund breed) in Nippon Veterinary and Animal Science University. All extruded disc samples showed hypertrophic changes and clustering of cells, typical features observed in the degenerated nucleus pulposus. The relative expression of type X collagen in the degenerated nucleus pulposus (84.3 +/- 11.0%) was significantly increased compared to the control nucleus pulposus (5.4 +/- 5.4%). Our findings suggest that type X collagen might contribute to the development of degeneration or calcification in the nucleus pulposus of the chondrodystrophoid dog.

Fluconazole (Fcz) is successfully used in human organ transplant patients as an antifungal therapy. However, Fcz can increase the cyclosporine (CsA) trough level and lead to CsA nephrotoxicity. In canine renal transplantation, CsA has been used as a major immunosuppressant, and it is important to control its trough level. However, the interaction of Fcz with CsA has not yet been reported in dogs. In this study, the effect of Fcz treatment on the pharmacokinetics of CsA in four healthy beagles was investigated using a four-period crossover design. The treatments included CsA alone (A), CsA + multiple-dose Fcz 50 mg (B), CsA + multiple-dose Fcz 25 mg (C) and CsA + single-dose Fcz 50 mg (D). Blood CsA concentrations were measured at 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hr after CsA administration. The AUC(0-12) and C(max) values for treatment B were significantly higher than those for the other treatments. In particular, the AUC(0-12) of treatment B was about two times higher than that of treatment A. Fcz administration did not significantly prolong the half-life or mean residence time of CsA. The results of our study show that administration of multiple therapeutic doses of Fcz can significantly increase the CsA blood concentration, which might partially depend upon the Fcz blood concentration. When Fcz is used in CsA-based canine renal transplantation, it may be necessary to adjust the CsA trough level by decreasing the dose.

The purpose of this study was to determine factors correlated with the severity of radiographic osteoarthritis (OA) scoring in dogs with cranial cruciate ligament rupture (CrCLR). Three radiographs of stifle joints (craniocaudal, mediolateral, and mediolateral radiograph with 90 degree flexion of the stifle and tarsal joints) were obtained from 36 dogs with CrCLR (Clinical group) and from 22 dogs without stifle joint disease (Control group). Information about these dogs was collected from the owners and from medical records. Radiographic OA scores in each dog in the clinical group were determined from radiographs using a numeric grading system previously reported. The tibial plateau angle (TPA) in each dog in both groups was measured on mediolateral radiographs with 90 degree flexion of the stifle and tarsal joints. The Mann-Whitney's U test was used for comparing variables between the clinical group and the control group, and Spearman's rank correlation test was used for evaluating correlations between radiographic OA scores and variables in the clinical group. No significant differences were detected between the clinical group and the control group for any of the variables. There were two positive correlations; one between the radiographic OA score and TPA (r=0.395, p=0.014); and the other between body weight and OA score (r=0.399, p=0.013) in the clinical group. Our results indicate that body weight and TPA could affect the severity of the radiographic OA score in dogs with CrCLR.<br>

Objective To measure and compare activities of interleukin-1 beta (IL-1 beta), 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-1 beta, 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-1 beta 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. (c) Copyright 2006 by The American College of Veterinary Surgeons.

Acute diabetes insipidus-like symptoms have been reported as a complication after hypophysectomy in dogs. These symptoms are believed to be the consequence of deficiency of arginine vasopressin (AVP) secretion. The symptoms spontaneously resolve within 2 weeks, but the mechanism is unclear. In the present study, AVP secretion related to increases in Na⁺ concentration and serum osmotic pressure was measured, and immunohistochemical analysis in the paraventricular and supraoptic nuclei was perfomed after hypophysectomy in normal dog. In the hypertonic saline test, the plasma AVP concentration slightly increased in hypophysectomized dogs, although the increase was markedly smaller than that in normal dogs. An immunohistochemical study of the hypothalamus nucleus revealed that, AVP-positive cells tended to decrease after hypophysectomy. It suggests that excision of the posterior lobe by surgery injured the axon of magnocellular neuron in the hypothalamus. A decrease in the function and the number of AVP-producing and -secreting magnocellular neurons after hypophysectomy, suggests that the clinical improvement of postoperative diabetes insipidus-like symptoms may not be related to the recovery of AVP secretion.<br>

Objective-To compare activities of interleukin (IL)-1 beta, IL-6, tumor necrosis factor (TNF)-alpha, and matrix metalloproteinase (MMP)-3 and contents of sulfated glycosaminoglycan (S-GAG) in joint fluid obtained from dogs with hip dysplasia (HD) and clinically normal dogs, evaluate correlations among these markers in joint fluid obtained from dogs with HD, and evaluate correlations between each marker and clinical and radiographic variables. Animals-26 dogs with HD (clinical group) and 43 clinically normal Beagles (control group). Procedure-Joint fluid was aseptically collected from the hip joints of all dogs. For each dog in the clinical group, age, duration of lameness, radiographic osteoarthritis (OA) score, and Norberg angle in each affected joint were recorded. Activities of IL-1 beta, IL-6, TNF-alpha, and MMP-3 and S-GAG contents were measured. Values were compared between groups by use of Mann-Whitney U tests, and the Spearman rank correlation test was used to evaluate correlations among markers and between each marker and clinical or radiographic variables. Results-Values of all markers were significantly higher for the clinical group, compared with values for the control group. There was a moderate positive correlation between lameness duration and IL-6 activity and a strong negative correlation between the Norberg angle and IL-1 beta activity. Conclusions and Clinical Relevance-Analysis of our results indicated that there was a significant increase in markers of OA in dogs with HD. Activities of IL-1 beta and IL-6 in joint fluid of dogs with HD may be influenced by the severity of laxity in the hip joint and lameness duration, respectively.

Objective-To determine the effects of continuous low-dose infusion of lipopolysaccharide (LPS) on the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) mRNA and neutrophil accumulation in the lungs, liver, spleen, small intestine, and pancreas in dogs. Animals-11 healthy adult Beagles. Procedure-Dogs received a continuous infusion of a low dose (10 mu g/kg/h, IV) of LPS (Escherichia coli 055:135) or saline (0.9% NaCl) solution (20 mL/kg/h, IV) for 8 hours. Activity levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) and the number of WBCs in circulation were examined before and 1, 2, 4, and 8 hours after the onset of LPS infusion. Expression of E-selectin and ICAM-1 mRNA and the number of neutrophils in each tissue were examined. Results-After the onset of LPS infusion, serum TNF-alpha and IL-1 beta activities transiently increased. Thereafter, IL-6 activity increased, and high IL-6 activity was maintained throughout the experiment. In dogs in the LPS group, expression of E-selectin mRNA increased only in the lungs, and expression of ICAM-1 mRNA increased in the lungs and liver; the number of neutrophils in the tissue increased in the lungs and liver. Conclusions and Clinical Relevance-Results suggested that expression of E-selectin and ICAM-1 mRNA increased during sepsis, particularly in the lungs and liver, and that this increase was associated with neutrophil accumulation. Hence, inhibiting the activation of endothelial cells in the lung and liver may decrease organ damage caused by accumulated neutrophils and help regulate multiple-organ dysfunction.

Purpose. Recently, MMP-7 and MMP-3 have been found to play a crucial role in the natural resorption process of herniated discs. We therefore examined the role of these recombinant human matrix metalloproteinases (rh MMPs) in the treatment of herniated discs. Methods. (a) Surgical samples of herniated disc were cultured in the presence or absence of rh MMPs, and wet weight was measured 24h later. (b) The rh MMPs were administered into normal rabbit intervertebral discs, and after 1 week spine samples were stained with Safranin O. (c) The rh MMPs were administered into canine herniated discs in vivo. Myelography and MRI were performed prior to and 1 week after administration. Spine samples were examined histologically. Whole disc tissue was collected, total protein was extracted, and Western blot analysis was performed. Results. (a) Proteoglycan degradation was found in MMP-7, MMP-3, and chymopapain-treated samples. MMP-7 and chymopapain-treated samples displayed a significant loss in wet weight (p &lt 0.01). (b) Normal disc tissues after administration of rh MMP-7, MMP-3, and chymopapain showed an extensive loss of Safranin O staining. (c) The rh MMP-7-treated discs had a marked decrease in protruded herniation by MRI. Herniated discs after administration of MMP-7 and chymopapain showed a significant decrease in protruded mass 7 days after administration compared with saline-treated discs when evaluated by myelography (p &lt 0.01). The rh MMP-7-treated discs displayed a clear loss of Safranin O staining in the nucleus pulposus. Proteoglycan expression was barely detectable in disc tissues after MMP-7 administration, whereas obvious expression was obtained in saline-treated or untreated disc tissues. Conclusions. Exposure to rh MMP-7 resulted in promising proteoglycan loss in human surgical samples, normal rabbit intervertebral discs, and natural canine herniated discs. Administration of rh MMP-7 may facilitate the resorption process of herniated discs. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Objective-To determine whether continuous infusion of a low dose of lipopolysaccharide (LPS) to induce a condition mimicking septic shock in dogs would affect systemic and hepatosplanchnic circulation and oxygenation. Animals-12 healthy adult Beagles. Procedure-Dogs received a low dose of LPS (Escherichia coli 055:135) by continuous IV infusion at a rate of 1 mug/kg/h for 8 hours. Systemic hemodynamics; systemic oxygenation;, blood flow in the cranial mesenteric artery, common hepatic artery, and portal vein; intestinal and hepatic tissue blood flow; mesenteric oxygenation; and intramucosal PCO2 were examined before and at selected time points after onset of the LPS infusion. Results-After onset of the LPS infusion, cardiac index increased and mean arterial pressure (MAP) and systemic vascular resistance decreased, which is characteristic of the hyperdynamic state in septic patients: Hepatosplanchnic blood flow increased during the hyperdynamic state. Intestinal PCO2 was increased even when blood flows increased. During the latter half of the experimental period, MAP was maintained but hepatosplanchnic blood flows decreased and intestinal PCO2 increased further. Conclusions and Clinical Relevance-Analysis of the results suggested that hepatosplanchnic blood flow enters the hyperdynamic state during the early stages of sepsis and that intestinal tissue oxygenation is threatened even when hepatosplanchnic blood flow is increased or maintained. Hence, improvement of hepatosplanchnic circulation and intestinal tissue oxygenation is important in dogs with clinical evidence of a septic condition.

A shock model was experimentally produced by intravenous injection of a lethal dose (3 mg/kg) of endotoxin under general anesthesia induced by pentobarbital sodium using 7 beagles. The effect of this endotoxic shock on the reticuloendothelial function was investigated. The blood endotoxin concentration peaked immediately after administration and decreased subsequently. However, the value still remained on an increased level (1,051 pg/ml) even at 360 min after endotoxin treatment. The lipid emulsion test as an index of reticuloendothelial phagocytotic activity and the arterial ketone body ratio as an index of the energy charge in the liver decreased after endotoxin treatment and failed to recover during the experiment. Fibronectin, one of opsonic proteins, tended to decrease after injection of the endotoxin and was significantly (p&lt;0.01) low at 180 and 360 min compared with the value before injection of the endotoxin. These results suggested the depression of the reticuloendothelial function during endotoxin-induced shock.