Bone morphogenetic protein 4 and bone morphogenetic protein receptor expression in the pituitary gland of adult dogs in healthy condition and with ACTH-secreting pituitary adenoma.

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.

A magnetic resonance imaging-based classification system for indication of trans-sphenoidal hypophysectomy in canine pituitary-dependent hypercortisolism.

OBJECTIVES: The objectives of this study were to establish a magnetic resonance imaging-based classification system for canine hyperadrenocorticism according to pituitary gland extension, determine indications for trans-sphenoidal hypophysectomy, and clarify the prognosis for each disease grade. METHODS: A 5-point classification system (Grades 1 to 5) was developed based on tumour extension in dorsal and cranio-caudal directions. Cases were then classified as Type A: no arterial circle of Willis or cavernous sinus involvement and Type B: cases in which these blood vessels were involved. RESULTS: Medical records and magnetic resonance imaging data of 37 cases with hyperadrenocorticism were reviewed. Thirty-three cases underwent surgery; 4 Grade 5 cases did not have appropriate indications for surgery, and other therapies were used. Complete resection was achieved for 3, 3, 22 and 1 Grade 1A, 2A, 3A and 3B cases, respectively. Resection was incomplete in 1, 1 and 2 Grade 3A, 3B and 4B cases, respectively. Remission was achieved in 29 cases. Recurrence occurred in 4 cases, all of which were classified as Grade 3. CLINICAL SIGNIFICANCE: Dogs with Type A, Grade 1 to 3 hyperadrenocorticism had a good prognosis following trans-sphenoidal hypophysectomy. Grade 3B, 4 and 5 cases may not be suitable for this surgery.

Histological and immunohistological analysis of degenerative changes in the cranial cruciate ligament in a canine model of excessive tibial plateau angle.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CLINICAL SIGNIFICANCE: 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.

Tibial displacement with stifle joint flexion and cranial cruciate ligament transection in the dog. An ex vivo study using a robotic simulator.

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°.

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.

Marked depletion of the water-channel protein, AQP5, in the canine nictitating membrane glands might contribute to the development of KCS.

The objectives of this study were to investigate the normal histological localization of aquaporin (AQP) 5 protein in the lacrimal and nictitating membrane glands and to compare this localization in healthy and keratoconjunctivitis sicca (KCS) dogs. Lacrimal and nictitating membrane glands of 5 healthy Beagles and nictitating membrane glands of 5 KCS dogs (3 Beagles and 2 mongrel dogs: 0-13 years) were used for the present study. The owners of the KCS dogs did not consent to perform biopsies of the lacrimal glands. The localization and distribution of AQP5 protein were investigated by an immunohistochemical technique. In immunohistochemical staining, AQP5 was localized in the apical site of acinar epithelial and ductal epithelial cells from both the lacrimal and nictitating membrane glands in healthy dogs. However, AQP5 was not detected in the 5 KCS dogs. These results for immunohistochemical AQP5 localization might correlate with the deficiency in tear secretion found in KCS dogs.

Role of an ATP-sensitive potassium channel opener, YM934, in mitochondrial energy production in ischemic/reperfused heart.

We examined a possible mechanism of action of an ATP-sensitive potassium (K(ATP)) channel opener, YM934, for the improvement of energy metabolism in hearts subjected to 35-min ischemia and 60-min reperfusion. The treatment with 30 nM YM934 for the final 15 min of preischemia enhanced postischemic recovery of left ventricular developed pressure, attenuated the postischemic rise in left ventricular end-diastolic pressure, and suppressed the release of creatine kinase and ATP metabolites during reperfusion. The treatment also restored myocardial ATP and creatine phosphate contents and attenuated the decrease in mitochondrial oxygen consumption rate during reperfusion. The higher mitochondrial function was also seen in YM934-treated hearts at the end of ischemia. In another set of experiments, myocardial skinned bundles were incubated for 30 min under hypoxic conditions in the presence and absence of YM934, and then mitochondrial oxygen consumption rate was determined. Hypoxia decreased the mitochondrial oxygen consumption rate of skinned bundles to approximately 40% of the prehypoxic value. In contrast, the treatment of skinned bundles with 30 nM YM934 preserved the mitochondrial oxygen consumption rate during hypoxia. The effect of YM934 on the hypoxic skinned bundles was abolished by combined treatment with either the K(ATP) channel blocker glyburide or the mitochondrial K(ATP) channel blocker 5-hydroxydecanoate in a concentration-dependent manner. The results suggest that YM934 is capable of attenuating ischemia/reperfusion injury of isolated perfused hearts due to preservation of mitochondrial function during ischemia, probably through opening of mitochondrial K(ATP) channels.