Relationship between clinical parameters and malformations in dogs diagnosed with atlanto-axial instability.

Atlanto-axial instability is a common disease that affects toy-breed dogs. Most cases of atlanto-axial instability are congenital. Furthermore, patients with atlanto-axial instability are predisposed to other concurrent diseases. Therefore, this study aimed to retrospectively determine the presence of concurrent diseases in cases with atlanto-axial instability using imaging data and analyze the relationship between clinical parameters and the incidence of complex malformations. The clinical data and imaging findings of 41 toy-breed dogs diagnosed with atlanto-axial instability were analyzed using their medical records and imaging data. Occipital dysplasia (17/27), atlanto-occipital overlapping (22/34), dens dysplasia (27/41), Chiari-like malformation (8/34), syringomyelia (5/34), lateral ventricular enlargement (20/36), and intracranial arachnoid cyst (5/35) were observed in patients with atlanto-axial instability. The body weight of the patients in the groups with atlanto-occipital overlapping and lateral ventricular enlargement was lower than that of those in the groups without these diseases (1.78 ± 0.71 vs 2.71 ± 1.15 kg, P = 0.0269, 1.60 ± 0.40 vs 2.75 ± 1.08 kg, P = 0.001, respectively). Furthermore, when the correlation between the total number of concurrent diseases and the age at onset and body weight was examined, it became clear that lower body weight was associated with the incidence of a greater number of concurrent diseases. Thus, the findings of this study suggest that toy-breed dogs are more likely to present with complex malformations and should be evaluated carefully with additional examinations and treatment methods.

Osteosarcoma of the hyoid bone in a cat.

Case summary: A 10-year-old male neutered Abyssinian cat was presented with the chief complaint of a right pharyngeal mass. On palpation, the mass was easily palpable; it appeared well demarcated and mobile, and oval in shape. Radiographic examination showed a radiopaque pharyngeal mass, which was revealed to be a well-demarcated mass lesion with a radiopaque marginal area on CT examination. The inside of the mass was uniformly well enhanced on contrast CT examination. A three-dimensional image reconstructed from the CT images strongly suggested that the mass originated from the right stylohyoid bone. No abnormal lesions were observed in the thoracic and abdominal CT examinations. Surgical excision of the mass was scheduled 3 weeks after the first admission. The mass was bluntly separated from the surrounding musculature and resected together with the ipsilateral epihyoid bone adjacent to the mass. Histopathological examination of the resected mass showed neoplastic cells with osteoid formation, and the mass was histopathologically diagnosed as an osteosarcoma. The postoperative recovery from anaesthesia was uneventful, and the cat began feeding on its own from the third postoperative day. The 3-month postoperative CT evaluation revealed no local recurrence or distant metastasis. The cat showed no abnormal findings at the time of writing (6 months postoperatively). Relevance and novel information: Although osteosarcoma derived from the hyoid bone has rarely been reported in the veterinary field, this report suggests that such tumours may occur in cats. In addition, partial excision of the hyoid apparatus seems to be well tolerated in cats.

Sulfasalazine, a potent cystine-glutamate transporter inhibitor, enhances osteogenic differentiation of canine adipose-derived stem cells.

Cystine-glutamate transporter (xCT) is a plasma membrane transporter that imports cystine and indirectly contributes to the oxidative stress resistance associated with increased intracellular glutathione levels. Canine adipose-derived stem cells (CADSCs) include an xCT-positive subpopulation and show relatively low expression of osteogenic markers during in vitro osteogenic differentiation. Sulfasalazine (SSZ), a drug used to treat rheumatoid arthritis, suppresses xCT expression in cancer cells. In this study, we found that the SSZ treatment at 100 µM significantly suppressed xCT mRNA expression in CADSCs but did not significantly affect cell proliferation under the same conditions. Additionally, this treatment decreased the intracellular glutathione concentration. During in vitro osteogenic differentiation, the SSZ treatment at 50 µM and 100 µM significantly increased alizarin red staining and its quantification, as well as the concentration-dependent osteogenic differentiation markers (BMP1 and SPP) mRNA expression. Our results suggested that SSZ enhances the osteogenic differentiation potential of CADSCs and can potentially exhibit a superior therapeutic profile in canine bone regenerative medicine.

Comparison of surgical invasiveness between micro-endoscopic discectomy/microscopic discectomy and conventional hemilaminectomy in dogs.

Micro-endoscopic discectomy (MED) or microscopic discectomy (MD) have been performed for disc herniation in humans. The purpose of this study was to compare the invasiveness of the hemilaminectomy in dogs between the approach using a cylindrical retractor for the MED/MD and a conventional open surgical approaches in dogs. First, as preliminary studies, we analyzed the suitability of the cylindrical retractor for the vertebral body of small to medium-sized dogs on the X-ray computed tomographic images using the three-dimensional analysis software, and confirmed that it was possible to open a bone window of an approximate length of 1.72 clto the spinal canal with the cylindrical retractor with a diameter 17 mm using two medium-sized canine cadavers. Next, to determine difference in the invasiveness of hemilaminectomy, the magnitude of tissue damage, surgical stress and postoperative pain were compared between the conventional open approach (hemilaminectomy group: HL group, n=6) and the surgical approach using the cylindrical retractor (MD group, n=6) in 12 beagle dogs. The plasma creatine phosphokinase, C-reactive protein and cortisol concentrations, incision length and University of Melbourne Pain Scale scores after the hemilaminectomy were significantly lower in the MD group than in the HL group. There were no significant differences between the durations of surgery and the other evaluated indices. The approach using the MD can provide a less invasive hemilaminectomy than the conventional approach in dogs.

Histiocytic sarcoma with spinal necrosis in a dog with progressing non-ambulatory tetraparesis.

Background: Histiocytic sarcoma (HS) is an aggressive malignant neoplasm, and widespread metastasis occurs with a fatal outcome. HS involving the central nervous system is relatively uncommon. Spinal cord necrosis, a very rare condition, could be induced by ischemia or infarction. Here, we report a dog progressing non-ambulatory tetraparesis with spinal cord necrosis caused by HS. Case Description: A 9-year-old male Labrador Retriever was presented with a progressing non-ambulatory tetraparesis. CT imaging revealed lysis of the spinous process of T7 and a ring-shaped lesion surrounding the soft tissue of lung fields. T2-weighted MRI showed the spinous processes of T6 to T8 as hyperintense, and the lesion infiltrated into the T7 vertebra and the spinal cord. After euthanasia, the final diagnosis upon necropsy was HS, which was observed in the lung, spinous process, thoracic cord, and the pulmonary hilar lymph node. Moreover, necrotic spots were spread widely through the thoracic spinal cord. Conclusion: This report outlines a case of canine HS in the lung, spinous process, thoracic cord, and pulmonary hilar lymph node. Ischemic deficit and necrosis of the thoracic spinal cord resulted from the compression of perivascular tumor cells, which rapidly led to progressive tetraparesis. Although the diagnosis was difficult, MRI and CT images helped determine the prognosis. To our knowledge, this is the first case report of canine HS with direct spinal cord involvement associated with spinal necrosis.

A Monte Carlo study on dose distribution of an orthovoltage radiation therapy system.

It is important to plan radiotherapy treatment and establish optimal dose distribution to reduce the chances of side effects and injury. Because there are no commercially available tools for calculating dose distribution in orthovoltage radiotherapy in companion animals, we developed an algorithm to accomplish this and verified its characteristics using tumor disease cases. First, we used the Monte Carlo method to develop an algorithm to calculate the dose distribution of orthovoltage radiotherapy (280 kVp; MBR-320, Hitachi Medical Corporation, Tokyo, Japan) using BEAMnrc at our clinic. Using development of Monte Carlo method, dose distribution for tumor and normal organs were evaluated in brain tumors, squamous cell carcinomas of the head, and feline nasal lymphomas. In all cases of brain tumors, the mean dose delivered to the GTV ranged from 36.2 to 76.1% of the prescribed dose due to the decrease through the skull. In the nasal lymphoma in cats, the eyes with covered a 2 mm-thick lead plate, the respective average dose to the eyes was 71.8% and 89.9% less than that to the uncovered eyes. The findings may be useful for informed decision making in orthovoltage radiotherapy with more effective and targeted irradiation and data collection allowing detailed informed consent.

Establishment of a New Canine Inflammatory Mammary Carcinoma Cell Line and Analysis of its Cystine-glutamate Transporter Subunit Expression.

Introduction: Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan. Material and Methods: The patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated. Results: The cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified. Conclusion: A new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

Outcomes of megavoltage radiotherapy for canine intranasal tumors and its relationship to clinical stages.

Background: Radiation therapy is considered important for the treatment of intranasal tumors in dogs and is believed to be essential for prolonging their survival. Aim: To investigate the contribution of clinical staging to improve outcomes of megavoltage radiotherapy for canine intranasal tumors. Methods: A total of 123 dogs with intranasal tumors were included in the study. Forty-eight dogs received orthovoltage radiotherapy after cytoreductive surgery (Group I), 21 received orthovoltage radiotherapy without surgery (Group II), and 54 received megavoltage radiotherapy without surgery (Group III). All cases in each group were classified into clinical stages 1-4, and the median survival time (MST) was compared for each stage in all groups. Results: The overall MST was not significantly difference among Group I (325 days), Group II (317 days), and Group III (488 days); however, Group III was prolonged than Groups I and II. The MSTs for stages 1, 2, 3, and 4 were 597, 361, 267, and 325 days in Group I; 633, 260, 233, and 329 days in Group II; and 931, 860, 368, and 176 days in Group III, respectively. The MST for stage 2 cases in Group III was significantly prolonged when compared with that in Groups I and II; no significant difference was observed at other stages; however, the MST in Group III was longer in stage 1. These results showed that megavoltage radiotherapy prolonged the MST in dogs with intranasal tumors when compared to orthovoltage radiation with or without cytoreductive surgery, and that improvements in MST at stage 2 contributed significantly to this. Conclusion: The improvement in the MST in dogs with stages 1 and 2 intranasal tumors highlights the importance of starting megavoltage radiotherapy in the early stages.

Adjustment of multi-leaf collimator parameters in 4-MV and 6-MV IMRT: A study of veterinary clinical cases.

Background: For optimal treatment, it is important to maintain optimal multi-leaf collimator (MLC) transmission in intensity-modulated radiation therapy (IMRT). However, adjustment of transmissions has not been reported in veterinary medicine. Aim: To demonstrate that appropriate MLC parameter adjustment for IMRT using 4- and 6-MV energy can reduce the need for quality assurance revalidation in real companion animal clinical cases. Methods: The MLC parameters (leaf transmission and leaf offset) of the treatment planning system were adjusted by evaluating seven plans (10 × 10 cm, 3ABUT, DMLC, 7segA, FOURL, HDMLC, and HIMRT) and 20 preclinical cases (10 cases each in 4- and 6-MV groups). Subsequently, 101 IMRT plans of 88 cases (77 dogs and 11 cats) were evaluated for absolute dose of plan target volume (PTV) and organs at risk (OAR) and were analyzed for the relative dose distribution by gamma analysis (3%/3 mm, >10%) using EBT3 film. Results: After adjustment of the MLC parameters (leaf transmission and leaf offset, 4 MV: 0.008 and 0, 6 MV: 0.005 and 0, respectively), the data from 101 plans (4 MV: 64 plans and 6 MV: 37 plans) treated with IMRT showed PTV <3%, OAR <5%, and gamma analysis pass rates ≥95% in all cases. Conclusion: Clinically meaningful dose distributions can be created even with a limited validation device if the treatment parameters are adjusted appropriately, even for tumors in canines and felines, where the irradiation field is small, the target is adjacent to the OAR, and the target is often superficial.

A long survival case of spinal nephroblastoma in a dog.

Background: Dogs' nephroblastoma of the spinal cord is a rare neoplastic disease, with few reports of long-term survival after surgery. We experienced that surgical treatment with postoperative radiation therapy for spinal nephroblastoma in a dog resulted in the long-term survival of 11 years. Case Description: The patient presented to our veterinary hospital because of progressive hindlimb paralysis. Based on diagnostic imaging, she was diagnosed with a thoracolumbar spinal cord tumor and was treated with surgery. The gross tumor tissue was removed after laminectomy, followed by postoperative radiation therapy using orthovoltage equipment. The histopathological features of the surgical specimen were consistent with those of previously reported spinal nephroblastoma, although infrequent mitotic figures were observed. The dog recovered well after treatment and resumed her normal walking condition. No tumor recurrence was observed on periodic follow-up magnetic resonance imaging performed 10 and 21 months after surgery. Imaging evaluation for the gradual development of hindlimb weakness was performed 9 years after surgery; however, no recurrence of tumor tissue was observed, and spondylosis deformans, probably induced after laminectomy, were identified as a possible cause. The dog died of aspiration pneumonia 11 years after surgery, independent of spinal nephroblastoma. Conclusion: To date, no clinical cases of canine spinal cord primary nephroblastoma that survived for 11 years after surgery have been reported. This case strongly suggests that providing intensive treatment for canine spinal nephroblastoma is very important.

Effect of drug-eluting bead transarterial chemoembolization loaded with cisplatin on normal dogs.

Transcatheter arterial embolization (TAE) and transcatheter arterial chemoembolization (TACE) are standard treatments for advanced hepatocellular carcinoma (HCC) and particularly for unresectable tumors or liver metastases in humans. However, reports on TACE used in veterinary medicine are few. This study aimed to evaluate the feasibility and safety of drug-eluting bead transarterial chemoembolization (DEB-TACE). We performed DEB-TACE in four clinically normal dogs and pharmacokinetically compared the results against hepatic arterial infusion (HAI) of cisplatin in two dogs. Drug-eluting beads (DEB) loaded with cisplatin were injected through a microcatheter for selective embolization of the left hepatic artery. After embolization, computed tomography (CT) images and histological examination findings were obtained during a 4-week observation period. Serum platinum concentrations were measured to evaluate cisplatin after each procedure. Biochemical analysis was performed during a 12-week observation period. Embolization was successful in all dogs, and there were no clinically apparent abnormalities. Embolization was confirmed up to 4 weeks after DEB-TACE in two of the four dogs and up to 1 week in the other two dogs using postoperative CT images. Cisplatin was not detected in peripheral veins in all dogs after DEB-TACE, but it was detected in trace amounts after HAI. DEB-TACE using cisplatin was safe and well tolerated by normal dogs. DEB-TACE may be useful in terms of determining systemic toxicity and drug concentration within tumors.

Evaluation of setup errors of immobilization device for radiation therapy in companion animals.

Background: Intensity-modulated radiotherapy (IMRT), which allows generating steep dose gradients, is a beneficial treatment for companion animals with adjacent target and risk organs. IMRT is essential for high setup accuracy for avoiding overdose to risk organs, and optimal radiotherapy is important for evaluating the setup accuracy of companion animals. Aim: To use an immobilization device to evaluate setup errors in radiotherapy for companion animals. Methods: We calculated setup errors in radiotherapy for 386 animals (dogs and cats; 3,261 registration images) that underwent radiotherapy between 2016 and 2022. The companion animals were immobilized with a customized bite block and vacuum lock device. A quantile-quantile plot with 95% confidence interval (CI) was used to evaluate the histogram of the setup errors, and the systematic and random setup errors were calculated for each region (brain, head and neck, chest and abdomen, pelvis, and spine). Results: The setup error in each direction presented an extremely narrow-interval histogram, with the following lower and upper 95% CIs: cranial-caudal (-0.08, -0.06 cm); left-right (-0.04, -0.02 cm); and dorsal-ventral (-0.13, -0.11 cm). The mean systematic setup error was 0.16 cm (range: 0.12-0.36 cm), and the random error was 0.15 cm (range: 0.08-0.34 cm). The pelvis showed the highest systematic and random setup errors (mean: 0.36 and 0.23 cm, respectively). Conclusion: The use of an immobilization device enables highly accurate radiotherapy for companion animals (95% CI < 0.15 cm).

Sphere-forming cells display stem cell-like characteristics and increased xCT expression in a canine hepatocellular carcinoma cell line.

Hepatocellular carcinoma (HCC) is the most common type of canine primary liver tumor; however, most chemotherapies against HCC are ineffective due to resistance to anticancer agents. Sphere-forming cells are considered to act as cancer stem cells for various types of solid tumors and have been established for many canine tumor cell lines, yet no studies have reported sphere-forming cells for canine HCC. In this study, we established sphere-forming cells from a canine HCC cell line (AZACH). These cells displayed increased stem cell marker mRNA expression (Nanog, Sox2, c-Myc, and Klf4), aldehyde dehydrogenase activity, and chemoresistance against mitoxantrone, gemcitabine, and doxorubicin. In addition, sphere-forming cells exhibited higher xCT expression and lower intracellular reactive oxygen species levels than adherent cells. Treatment with sulfasalazine, a xCT-specific inhibitor, reduced sphere formation efficiency. Together, these results indicate that sphere-forming cells derived from a canine HCC cell line have similar characteristics to cancer stem cells and that their increased xCT expression and associated resistance to oxidative stress contribute toward sphere formation.

Clinical features and their course of pituitary carcinoma with distant metastasis in a dog.

An 11-year-old male toy poodle with neurological symptoms was diagnosed with a macroscopic pituitary tumor, which produced adrenocorticotropic hormone. Radiation therapy with a linear accelerator was performed for the pituitary tumor, and resulted in good local tumor control. However, serum endogenous adrenocorticotropic hormone concentrations were uncontrollable even after the tumor disappeared. Abdominal computed tomography revealed splenic masses, and splenectomy was performed. Histopathological examination of the surgical specimen showed tumor cells with eosinophilic and finely granular cytoplasm suggestive of endocrine origin. Since these cells were positive for adrenocorticotropic hormone, the case was diagnosed as a pituitary carcinoma with distant metastasis. Necropsy revealed multiple metastases to the abdominal organs. This is the first case report describing canine pituitary carcinoma with distant metastasis.

Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells.

Adipose-derived stem cells (ADSCs) are promising cell sources for regenerative medicine due to the simplicity of their harvest and culture; however, their biological properties are not completely understood. Moreover, recent murine and human studies identified several functional subpopulations of ADSCs varying in differentiation potential; however, there is a lack of research on canine ADSCs. Cystine transporter (xCT) is a stem cell marker in gastric and colon cancers that interacts with CD44 to enhance cystine uptake from the cell surface and subsequently accelerates intercellular glutathione levels. In this study, we identified a ~5% functional subpopulation of canine ADSCs with xCT+ expression (xCTHi). Compared with those of the xCT- subpopulation (xCTLo), the xCTHi subpopulation showed a significantly higher proliferation rate, higher expression of conventional stem cell markers (SOX2, KLF4, and c-Myc), and higher expression of adipogenic markers (FABP4 and PPARγ). By contrast, the xCTLo subpopulation showed significantly higher expression of osteogenic markers (BMP1 and SPP) than xCTHi cells. These results suggest xCT as a candidate marker for detecting a functional subpopulation of canine ADSCs. Mechanistically, xCT could increase the adipogenic potential while decreasing the osteogenic differentiation potential, which could serve as a valuable target marker in regenerative veterinary medicine.

Effect of transcatheter arterial embolisation in normal canine liver using trisacryl gelatine microspheres (Embosphere).

The effect of selective transcatheter arterial embolisation (TAE) using trisacryl gelatine microspheres (TGMs) in the normal canine liver was investigated. Selective embolisation was achieved by injecting TGMs into the left hepatic artery through a microcatheter in four healthy dogs. After embolisation, computed tomography (CT), biochemical analysis and histological examination were performed during a 12-week observation period. Embolisation was successful in all four dogs. Postoperative CT revealed consistent embolisation of the artery within the experimental period in three dogs. Hepatic enzyme levels slightly increased after embolisation but tapered to normal ranges. Histological examinations revealed no abnormal changes. Thus, selective TAE with TGMs was well tolerated in normal dogs and may be applicable to canine hepatocellular carcinoma.

Comparison of postoperative pain and inflammation reaction in dogs undergoing preventive laparoscopic-assisted and incisional gastropexy.

This study compared the effects of postoperative pain and inflammation reaction after preventive laparoscopic-assisted gastropexy (LAG) and incisional gastropexy (IG) in 10 clinically normal Beagles. Surgical time, incision length, visual analog scale (VAS) score, University of Melbourne Pain Scale (UMPS) score, and plasma C-reactive protein (CRP), plasma cortisol (COR), and serum interleukin-6 (IL-6) levels were evaluated. The VAS and UMPS scores and COR and IL-6 levels were recorded at 0.5, 1, 2, 4, 8, 12, 18 and 24 hr after surgery. CRP level was recorded at 12, 24 and 48 hr after surgery. The VAS and UMPS scores showed no significant intergroup differences. Compared to IG, LAG had significantly lower surgical time (45 ± 9.91 min vs 64 ± 5.30 min; P<0.05), incision length (46 ± 8.21 mm vs 129 ± 19.49 mm; P<0.05), CRP level (12 hr after surgery; 4.58 ± 1.58 mg/dl vs 12.4 ± 1.34 mg/dl; P<0.01), and COR level (1 hr after surgery; 10.79 ± 3.07 µg/dl vs 15.9 ± 3.77 µg/dl; P<0.05). IL-6 levels showed no significant intergroup differences at any time point. However, LAG resulted in lower IL-6 levels than did IG at all postoperative time points. Neither procedure resulted in significant surgical complications. LAG produced lower surgical stress than did IG, suggesting that LAG is a safe, minimally invasive, and highly useful technique for preventing canine gastric dilatation-volvulus. Nevertheless, since this study used experimental models, its usefulness should be evaluated in future cases.

Identification of rhodamine 123-positive stem cell subpopulations in canine hepatocellular carcinoma cells.

The majority of cases of chemotherapy for hepatocellular carcinoma (HCC) are not effective in human or veterinary medicine due to resistance against anticancer agents. In human medicine, hepatocellular carcinoma stem cells (HCSCs) were recently identified as cytokeratin 19 (CK19)-, cluster of differentiation (CD)-44-, and CD133-positive. However, there are few previous reports regarding canine HCSC (cHCSC). Additionally, to the best of our knowledge, the chemoresistance against anticancer agents of these cHCSCs has not been investigated. In the present study staining of cHCSCs was performed with rhodamine 123, a low-toxicity fluorescent dye for mitochondria, by flow cytometry. There were two subpopulations in the HCC cell line defined by their higher (RhoHi) and lower (RhoLo) fluorescence intensity of rhodamine 123. The RhoHi subpopulation demonstrated a higher Nanog gene expression, sphere-forming ability, and chemoresistance against gemcitabine. However, there was no significant difference between RhoHi and RhoLo regarding the proliferation rate and chemoresistance against mitoxantrone and doxorubicin. The present results indicate that the expression of rhodamine 123 identifies different stem cell subpopulations in a canine HCC cell line.