MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases.

Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma.

Chemically modified synthetic microRNA-205 inhibits the growth of melanoma cells in vitro and in vivo.

microRNA (miR)-205 is downregulated and acts as a tumor suppressor in human melanoma cells. Previously, for clinical application, we added aromatic benzene-pyridine (BP-type) analogs to the 3'-overhang region of the RNA-strand and changed the sequences of the passenger strand in the miR-143 duplex. Here, we demonstrated the antitumor effect in vitro and in vivo of miR-205 that was also chemically modified by BP and had altered passenger sequence. In in vitro experiments, transfection with the synthetic miR-205 (miR-205BP/S3) significantly inhibited the growth of human melanoma cells. Exogenous miR-205BP/S3 suppressed the protein expression levels of E2F1 and VEGF, which are validated targets of miR-205-5p, and BCL2, a transcribed molecule of E2F1, as did Pre-miR-205, used as a miR-205 mimic having the wild-type sequence. On the basis of the results of a luciferase activity assay, miR-205BP/S3 directly targeted E2F1, as did Pre-miR-205. However, miR-205BP/S3 was much more resistant to RNase than Pre-miR-205 in fetal bovine serum and to RNase in mice xenografted with human melanoma tissues. In addition, the intratumoral injection of miR-205BP/S3 exhibited a significant antitumor effect compared with the case of control miRNA or Pre-miR-205 in human melanoma cell-xenografted mice. These findings indicate that miR-205BP/S3 is a possible promising therapeutic modality for melanoma.

Anti-oncogenic microRNA-203 induces senescence by targeting E2F3 protein in human melanoma cells.

MicroRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of their complementary mRNA. We recently reported that miR-203 is down-regulated, and its exogenous expression inhibits cell growth in canine oral malignant melanoma tissue specimens as well as in canine and human malignant melanoma cells. A microRNA target database predicted E2F3 and ZBP-89 as putative targets of microRNA-203 (miR-203). The expression levels of E2F3a, E2F3b, and ZBP-89 were markedly up-regulated in human malignant melanoma Mewo cells compared with those in human epidermal melanocytes. miR-203 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequence of either E2F3 or ZBP-89 complementary to miR-203. The ectopic expression of miR-203 in melanoma cells reduced the levels of E2F3a, E2F3b, and ZBP-89 protein expression. At the same time, miR-203 induced cell cycle arrest and senescence phenotypes, such as elevated expression of hypophosphorylated retinoblastoma and other markers for senescence. Silencing of E2F3, but not of ZBP-89, inhibited cell growth and induced cell cycle arrest and senescence. These results demonstrate a novel role for miR-203 as a tumor suppressor acting by inducing senescence in melanoma cells.

Synthetic microRNA-205 exhibited tumour suppression in spontaneous canine malignant melanoma by intratumoral injection.

MicroRNAs (miRNA) are small, noncoding RNA molecules consisting of 18 to 25 nucleotides. Malignant melanomas (MMs) are one of the most common malignancies in both dogs and humans. We previously reported that chemically modified synthetic miRNA-205 (miR-205BP/S3) inhibits melanoma growth in vitro and in vivo. The present study aimed to evaluate the efficacy of intratumoral administration of synthetic miR-205 for spontaneous CMMs and to evaluate its potential as systemic therapy. Ten dogs with various stages of MM were treated with miR-205BP/S3 injected into tumours. Adverse effects (AEs) were assessed in accordance with the Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines. Five cases attained complete remission (CR), three attained stable disease (SD), and two cases displayed characteristics of progressive disease (PD). In all cases, no changes were observed in the blood parameters upon miRNA administration, and miR-205BP/S3 administration did not yield any side effects. The present results suggest that intratumoral administration of miR-205BP/S3 is a potentially applicable treatment for canine melanoma.

B-cell intestinal lymphoma with Mott cell differentiation in a 1-year-old miniature Dachshund.

A 1-year-old intact female miniature Dachshund was presented with hematochezia, vomiting, and diarrhea of more than 1-week duration. An abdominal mass was palpated, which at exploratory surgery was found to be a 7-cm-long thickened section of ileum. The thickened ileum was resected. Impression smears revealed numerous small- to medium-sized lymphocytes, with a smaller number of cells resembling Mott cells. The Mott-like cells contained multiple pale vacuoles that were positive for periodic acid-Schiff (PAS) in wet-fixed smears, consistent with Russell bodies. Histologic evaluation of the surgically excised ileum revealed 2 populations of neoplastic lymphoid cells. The majority were uniform medium-sized lymphocytes with hyperchromatic oval or round nuclei and inconspicuous nucleoli. The remaining cells resembled Mott cells, which contained several PAS-positive eosinophilic globules in the cytoplasm, occasionally compressing the nucleus. The majority of neoplastic cells stained positively for vimentin, CD20, CD79a, and Pax-5, but were negative for CD3 and lysozyme; 43.5% of cells stained positively for Ki-67. The Mott cells were strongly positive for immunoglobulin but were negative for Pax-5. Using electron microscopy, a homogenous substance of intermediate electron density was observed frequently in the cisternae of rough endoplasmic reticulum in the cytoplasm of the Mott cells, and rarely in the perinuclear cisternae of the lymphoid cells, corresponding to the site of immunoglobulin staining. Monoclonal rearrangement of immunoglobulin heavy-chain (IgH) gene was observed by PCR testing for lymphocyte-antigen receptor rearrangement. The morphologic features, immunophenotype, and IgH gene rearrangement verified the lymphoid cells were neoplastic (mature cell type) and had a B-cell phenotype, with evidence of immunoglobulin production and differentiation into Mott cells. This case was unusual because of the age of the dog and because most intestinal lymphomas are T-cell phenotype. The Mott cell morphology also differed from typical mature B-cell lymphoma types and may be a unique B-cell lymphoma variant.

A case of pulmonary toxoplasmosis resembling multiple lung metastases of nasal lymphoma in a cat receiving chemotherapy.

An 11-year-old cat presented with nasal discharge and lacrimation and was diagnosed with nasal lymphoma. Although the cat showed favorable progression after undergoing chemotherapy, CT imaging demonstrated enlarged pulmonary nodules caused by Toxoplasma gondii. Following the cessation of chemotherapy, the cat was prescribed clindamycin hydrochloride for toxoplasmosis treatment; however, the cat developed kidney lymphoma and died. No T. gondii organisms were observed in the whole body necropsy specimens. It is known that immunocompromised human patients, including those who undergo chemotherapy, are considered at risk for toxoplasmosis. However, the risk of developing toxoplasmosis in cats undergoing chemotherapy is currently unknown. Findings from this case report suggest that cats with chemotherapy-resistant pulmonary masses might have a T. gondii infection rather than metastatic disease.

Short-Term Administration of Single-Agent Toceranib in Six Cases of Inoperable Massive Canine Hepatocellular Carcinoma.

Six dogs with massive hepatocellular carcinoma that was not amenable to surgery were treated by oral administration of single-agent toceranib at a dose of 2.0-3.0 mg/kg every other day for a minimum of 60 days. Partial response was achieved in three dogs, stable disease was achieved in one dog, and progressive disease occurred in two dogs, according to the canine Response Evaluation Criteria in Solid Tumors v1.0. Observed adverse events were mild to moderate in severity and reported in accordance with the Veterinary Cooperative Oncology Group's common terminology criteria for adverse events v1.1. Activities of alanine aminotransferase and alkaline phosphatase decreased in the cases that were sensitive to treatment with toceranib, whereas the activities remained high in resistant cases. Additionally, the level of phospho-vascular endothelial growth factor receptor 2 was found to be increased in a resistant case. Single-agent toceranib might prove to be an effective treatment for canine hepatocellular carcinoma pending further validation.

The tyrosine kinase inhibitor imatinib [STI571] induces regression of xenografted canine mast cell tumors in SCID mice.

Canine mast cell tumors (MCTs) are the most common cutaneous tumors in the dog. They have a wide range of behaviour, which can make these tumors challenging to treat. Recently, mutations in c-kit proto-oncogene have been identified in several canine MCTs. Imatinib is the first member of a new class of agents that act by inhibiting particular tyrosin kinase enzymes, including KIT which is a product of the c-kit. In this study the efficacy of imatinib to reduce or abolish canine MCT [CMC-1] using xenografted MCT in severe combined immunodeficient [SCID] mice was evaluated. Imatinib was administered at doses of 200mg/kg and 100mg/kg once a day for one week. The antitumor responses in SCID mice with CMC-1 xenografts following treatment with imatinib were observed. Significant tumor regression occurred with 100mg/kg on days 7, 10, 14 and 21, and 200mg/kg on all days. Our results indicate that imatinib is effective against canine mast cell tumor in mouse xenograft models. Canine MCTs might be a potential target for imatinib therapy.

The significance of p53 and retinoblastoma pathways in canine hemangiosarcoma.

To investigate whether inactivation of the p53 and retinoblastoma (Rb) protein pathways contributes to the development of canine hemangiosarcoma (HSA), we examined immunohistochemically the expression of p53, Rb, phosphorylated Rb (phospho-Rb), p16, and cyclin D1 in 39 spontaneous canine HSAs and 10 hemangiomas. In addition, mutations in the p53 gene were analyzed by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and PCR direct sequencing; furthermore, we quantified cyclin D1 mRNA by semiquantitative real-time reverse transcription-PCR. Positive immunoreactivity for p53 was observed in 17.9% of HSAs. However, mutations were not detected in these cases. The labeling indices for Rb, phospho-Rb, and cyclin D1 were markedly higher in all HSAs than in hemangiomas. Of the 7 cases with cyclin D1-positive immunoreactivity, 4 overexpressed cyclin D1 mRNA (to a level more than 10-fold higher than that of GAPDH mRNA). The p16 protein was clearly detected in all hemangiomas; however, 82% of the neoplastic cells in HSA showed a loss of or low immunoreactivity. These results suggest that alteration of the p16-cyclin D1-Rb pathway, rather than the p53 pathway, may be associated with the pathogenesis of canine HSA.

Circulating microRNA-214 and -126 as potential biomarkers for canine neoplastic disease.

Circulating microRNAs in the blood may provide diagnostic and prognostic information about canine neoplastic diseases, and their profiles may be conserved between human and canine species. We performed RT-qPCR to obtain the profiles of circulating plasma microRNA-214 and -126 in total 181 cases of canine neoplastic diseases and healthy controls. MicroRNA-214 levels were high in 2 epithelial tumours (thyroid and mammary carcinomas) and 4 non-epithelial tumours (osteosarcoma, histiocytic sarcoma, chondrosarcoma, and hemangiosarcoma). In contrast, microRNA-126 levels were high in 6 epithelial tumours (mammary, hepatocellular, squamous cell, thyroid, transitional cell carcinomas, and adenocarcinoma) and 4 non-epithelial tumours (osteosarcoma, mast cell tumour, melanoma, and hemangiosarcoma). The diagnostic potential of microRNA-214 was relatively high in sarcomas, whereas that of microR-126 was high in most types of the tumours. MicroRNA-214 and -126 were prognostic predictors in 2 groups (adenocarcinoma and non-epithelial tumours except for osteosarcoma) and 3 groups (epithelial tumours, adenocarcinoma, and melanoma), respectively. Additionally, the microRNA levels did not show a strong correlation with the other clinical parameters. In conclusion, circulating microRNA-214 and -126 have the potential to be diagnostic and prognostic biomarkers for canine neoplastic diseases. Furthermore, their profiles may be key references as well for exploring novel biomarkers for human cancers.

Hypersensitivity of BRCA2 deficient cells to rosemary extract explained by weak PARP inhibitory activity.

Rosemary extract is used in food additives and traditional medicine and has been observed to contain anti-tumor activity. In this study, rosemary extract is hypothesized to induce synthetic lethality in BRCA2 deficient cells by PARP inhibition. Chinese hamster lung V79 cells and its mutant cell lines, V-C8 (BRCA2 deficient) and V-C8 with BRCA2 gene correction were used. Rosemary extract and its major constituent chemicals were tested for their cytotoxicity by colony formation assay in cells of different BRCA2 status. The latter chemicals were tested for inhibitory effect of poly (ADP-ribose) polymerase (PARP) activity in vitro and in vivo. Rosemary has shown selective cytotoxicity against V-C8 cells (IC50 17 µg/ml) compared to V79 cells (IC50 26 µg/ml). Among tested chemicals, gallic acid and carnosic acid showed selective cytotoxicity to V-C8 cells along with PARP inhibitory effects. Carnosol showed comparative PARP inhibitory effects at 100 µM compared to carnosic acid and gallic acid, but the selective cytotoxicity was not observed. In conclusion, we predict that within rosemary extract two specific constituent components; gallic acid and carnosic acid were the cause for the synthetic lethality.

Computed Tomographic Evaluation of Presumptively Normal Canine Sternal Lymph Nodes.

The sternal lymph nodes receive drainage from a wide variety of structures in the thoraco-abdominal region. Evaluation of these lymph nodes is essential, especially in cancer patients. Computed tomography (CT) can detect sternal lymph nodes more accurately than radiography or ultrasonography, and the criteria of the sternal lymphadenopathy are unknown. The purpose of this retrospective study was to describe the CT characteristics of the sternal lymph nodes in dogs considered unlikely to have lymphadenopathy. The ratio of the short axis dimension of the sternal lymph nodes to the thickness of the second sternebra was also investigated. At least one sternal lymph node was identified in each of the 152 dogs included in the study. The mean long axis and short axis dimensions were 0.700 cm and 0.368 cm, respectively. The mean ratio of the sternal lymph nodes to the second sternebrae was 0.457, and the 95% prediction interval ranged from 0.317 to 0.596 (almost a fixed value independent of body weight). These findings will be useful when evaluating sternal lymphadenopathy using CT.

B-cell lymphoma with Mott cell differentiation in a cat.

A 12-year-old, male castrated Domestic Shorthair cat was presented to Animal Medical Center of Gifu Univeristy with anorexia and vomiting. Physical examination revealed an enlarged left tonsil and right mandibular lymph node (approximately 2-3× the normal size), and a submucosal mass on the right side of the epiglottis (1.5 × 2.0 cm). On computed tomography images, an enlarged left tonsil, and enlarged right mandibular, right pharyngeal, and left and right cervical lymph nodes were observed. Cytologic examination of smears of tonsil and lymph nodes revealed numerous medium- to large-sized neoplastic lymphoid cells, approximately half of which contained one or several light-blue homogenous globoid cytoplasmic inclusions (5-10 µm), which stained magenta with periodic acid-Schiff (PAS) stain. Histopathologic examination of the left tonsil revealed diffuse proliferation of medium- to large-sized neoplastic lymphoid cells effacing the original lymphoid architecture. Half of the cells contained one or several eosinophilic globoid cytoplasmic inclusions, which stained magenta with PAS and showed positive immunohistochemical reactions for immunoglobulin M (IgM) and λ light chain. Neoplastic lymphoid cells were also CD20+ , Pax5+ , and MUM1+ , and CD3- . Thus, the neoplastic lymphoid cells expressed a B-cell immunophenotype, and the globoid cytoplasmic inclusions represented an aberrant IgM λ light chain accumulation, similar to Russell bodies. B-cell lymphoma with Mott cell differentiation was diagnosed based on cytologic, histopathologic, and immunohistochemical features. This is the first report of B-cell lymphoma with Mott cell differentiation in a cat.

Canine tumor development and crude incidence of tumors by breed based on domestic dogs in Gifu prefecture.

We analyzed the status of tumor development in dogs by breed based on tumor cases that presented to the Department of Veterinary Pathology of the Gifu University for diagnostic examinations over eight years (2005-2012). We also calculated the crude incidence of tumors in dogs by breed based on the results of a survey conducted in 2011 in Gifu Prefecture. The most common sites of tumor development included the skin, digestive organs and mammary glands. Smaller dogs showed a tendency to have a higher incidence of breast tumors. We thus identified dog breeds with a higher crude incidence of tumors (Bernese mountain dog, golden retriever, corgi, etc.) and those with a lower crude incidence of tumors (Pomeranian, poodle, Chihuahua, etc.). Unlike the current trends for domestic dogs in the US and Europe, Japan has a higher number of small dogs as pets; it is therefore necessary to develop a policy for canine cancer specific to Japan.

Percutaneous transcatheter coil embolization of a ventricular septal defect in a dog.

A 4-month-old male French Bulldog weighing 5.0 kg (11 lb) was referred for a heart murmur. A grade 3/6 systolic murmur was detected at the left heart base and a grade 4/6 systolic murmur was detected at the right heart base. By use of color-flow Doppler ultrasonography and cardiac catheterization, a diagnosis of supracristal ventricular septal defect (VSD) with accompanying aortic regurgitation was made. Percutaneous transcatheter coil embolization was used to close the VSD. Because residual shunt was detected via echocardiography after coil implantation, the residual shunt was followed periodically via echocardiography to detect spontaneous closure of the VSD. Volume overload in the left ventricle was detected in the dog 131 days after admission. Additional coils were placed 137 days after admission. Hemolysis resulting in hemoglobinuria was detected, but this complication was mild. In the dog of this report, results of coil occlusion for correction of VSD were promising. Thus, coil occlusion should be considered as an alternative treatment for VSD in dogs.

Surgical correction of a partial atrioventricular septal defect with a ventricular septal defect in a dog.

Echocardiography of a dog with a cardiac murmur revealed an ostium primum septal defect, a ventricular septal defect, and mitral valve malformation with regurgitation. The mitral valve and tricuspid valve were separated and displaced at the same level as the ventricular septum. The mitral valve had a cleft in the septal cusp. Cardiac catheterization and angiocardiography showed a left-to-right shunt and a "goose-neck sign," which indicated an elongated left ventricular outflow tract. The diagnosis of a partial atrioventricular septal defect with ventricular septal defect was made. Surgical correction was successfully performed under extracorpo-real circulation using a cardiopulmonary bypass system.

Outcomes of dogs undergoing radiotherapy for treatment of oral malignant melanoma: 111 cases (2006-2012).

OBJECTIVE: To evaluate the characteristics and outcomes of dogs with stage I, II, III, or IV oral malignant melanoma treated by various types of radiotherapy. DESIGN: Retrospective case series. ANIMALS: 111 dogs. PROCEDURES: Medical records of dogs with oral malignant melanoma treated by radiotherapy (with or without adjunctive treatments) at a veterinary medical center between July 2006 and December 2012 were reviewed. Information regarding signalment, tumor location, disease stage, treatment protocols, adverse effects, and survival time were obtained from medical records and by telephone follow-up. Associations between variables of interest and outcome were analyzed. RESULTS: Dogs received orthovoltage x-ray (n = 68), megavoltage x-ray (39), or electron beam (4) radiotherapy. Adjunctive treatments included debulking surgery (n = 18), chemotherapy (39), or both (27). Median survival times for dogs with stage I, II, III, and IV melanoma were 758 days (n = 19), 278 days (24), 163 days (37), and 80 days (31), respectively, and differed significantly between dogs with stage I disease and those with all other disease stages. Among dogs with stage III melanoma, risk of death was significantly higher in those that received orthovoltage x-ray treatment than in those that received megavoltage x-ray treatment. Severe (primary or secondary) adverse effects were identified in 9 dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Median survival time was significantly longer for dogs with stage I oral malignant melanoma than for dogs with more advanced disease at the time of staging. The staging system used may be a useful tool for prognosis prediction in dogs undergoing similar treatment protocols for oral malignant melanomas.

MicroRNA-214 Promotes Apoptosis in Canine Hemangiosarcoma by Targeting the COP1-p53 Axis.

MicroRNA-214 regulates both angiogenic function in endothelial cells and apoptosis in various cancers. However, the regulation and function of miR-214 is unclear in canine hemangiosarcoma, which is a spontaneous model of human angiosarcoma. The expression and functional roles of miR-214 in canine hemangiosarcoma were presently explored by performing miRNA TaqMan qRT-PCR and transfecting cells with synthetic microRNA. Here, we report that miR-214 was significantly down-regulated in the cell lines used and in clinical samples of canine hemangiosarcoma. Restoration of miR-214 expression reduced cell growth and induced apoptosis in canine hemangiosarcoma cell lines through transcriptional activation of p53-regulated genes although miR-214 had a slight effect of growth inhibition on normal endothelial cells. We identified COP1, which is a critical negative regulator of p53, as a novel direct target of miR-214. COP1 was overexpressed and the specific COP1 knockdown induced apoptosis through transcriptional activation of p53-regulated genes as well as did miR-214-transfection in HSA cell lines. Furthermore, p53 knockdown abolished the miR-214-COP1-mediated apoptosis; thus, miR-214 and COP1 regulated apoptosis through controlling p53 in HSA. In conclusion, miR-214 functioned as a tumor suppressor in canine hemangiosarcoma by inducing apoptosis through recovering the function of p53. miR-214 down-regulation and COP1 overexpression is likely to contribute to tumorigenesis of HSA. Therefore, targeting miR-214-COP1-p53 axis would possibly be a novel effective strategy for treatment of canine hemangiosarcoma and capable of being applied to the development of novel therapeutics for human angiosarcoma.

Effects of dopamine infusion on cardiac and renal blood flows in dogs.

In veterinary medicine, dopamine is currently being administered clinically by infusion for treatment of kidney disorders at low doses (< or = 3 microg/kg/min) and for assessment of hemodynamics at high doses (> or = 5 microg/kg/min). However, since high doses of dopamine cause peripheral vasoconstriction due to its effect on alpha adrenoceptors, high doses have no longer been recommended. The present study was conducted to explore possible regimens for the use of dopamine infusion in dogs. The regional (renal and cardiac) blood flow for 60 min was measured by using colored microspheres at three doses (3, 10 and 20 microg/kg/min) of dopamine infusion in healthy anesthetized mongrel dogs. The effects on kidney and peripheral hemodynamics at each dose and the resultant cardiac output, mean arterial blood pressure and total peripheral resistance were determined. Renal blood flow increased markedly at 3 microg/kg/min dopamine. Improvement in hemodynamics indicated by marked increase in cardiac blood flow, cardiac output and mean arterial blood pressure and decreased total peripheral resistance was observed at higher doses (10 and 20 microg/kg/min). At 10 microg/kg/min, in addition to the satisfactory increase in cardiac blood flow, there was also a stable satisfactory increase in renal blood flow. However, at 20 microg/kg/min, increased myocardial oxygen consumption (manifested by marked increased in cardiac output), arrythmia and irregular increase in renal blood flow were detected. This study suggests that the clinical use of dopamine infusion in dogs could be safely expanded to moderately higher doses.

Implantation of permanent transvenous endocardial pacemaker in a dog with atrioventricular block.

A 10-year old male mongrel dog was presented to the University Veterinary Teaching Hospital with a two-month history of episodic syncope. Twenty-four hr Holter electrocardiographic (ECG) recording revealed frequent episodes of advanced atrioventriculer block with long periods of ventricular asystole. The cause of syncope was determined to be Adams-Stokes syndrome exhibited bradyarrhythmia. After the animal failed to respond to medical therapy, permanent transvenous pacemaker implantation was performed. Postoperative Holter ECG showed 100 beat per min programmed pacemaker rhythm, which indicated successful capture of the artificial pacing. The dog recovered smoothly from the operation and syncopal episodes completely disappeared. Six months after the surgery, no complications were observed and the dog's quality of life has dramatically improved.