The effect of adipose-derived mesenchymal stem cells and chondrocytes with platelet-rich fibrin releasates augmentation by intra-articular injection on acute osteochondral defects in a rabbit model.

BACKGROUND: This study aimed to investigate the efficacy of adipose-derived mesenchymal stem cells (ADSCs), platelet-rich fibrin releasates (PRFr), and chondrocyte transplantation in rabbit acute osteochondral defects. METHODS: Thirty rabbits were randomly assigned to five groups: untreated controls; ADSCs alone; PRFr alone; PRFr + ADSCs; and PRFr + chondrocytes. The critical size osteochondral defects in right knee femoral condyles were injected intra-articularly according to the groups, as listed. The experimental rabbits received treatments once a week for two weeks postoperatively. All evaluations were conducted for 14 weeks following surgery, and the regenerated cartilages were assessed by gross inspection and histological examination. RESULTS: There were no complications encountered in any of the rabbits. The size of the defect decreased and the volume of repaired cartilage increased in the medial femoral condyles of the PRFr + ADSCs group. Relative to the ADSCs or PRFr group, histological examination demonstrated that the PRFr + ADSCs group had thicker hyaline cartilage-specific extracellular matrix. Grading scores revealed that PRFr + ADSCs injection had better matrix, cell distribution, and surface indices than other groups (P < 0.05). However, the histological scores reported for PRFr + chondrocytes on cartilage repair were similar to those of PRFr, and there were no significant between-group differences. CONCLUSIONS: These findings showed that intra-articular injections of PRFr + ADSCs into the knee can reduce cartilage defects by regenerating hyaline-like cartilage without complications. This approach may provide an alternative method for functional reconstruction of acute osteochondral defects with an unlimited source of cells and releasates.

Apparent diffusion coefficient value for a B-cell central nervous system lymphoma in a cat.

CASE SUMMARY: This report involves a 10-year-old male mixed-breed cat with a B-cell central nervous system (CNS) lymphoma. The cat presented with ataxia progressing to left hemiparesis. While haematological findings were normal, serum biochemistry showed a high creatine phosphokinase concentration. MRI revealed a homogeneously enhancing well-demarcated extra-axial lesion involving the region of the left lateral aperture with oedema in left flocculus and left medulla oblongata. On diffusion-weighted imaging, the lesion margins showed marked hyperintensity relative to the right cerebellar hemisphere. On an apparent diffusion coefficient map, the lesion appeared hypointense, with an apparent diffusion coefficient value of 0.57 ± 0.01 × 10-3 mm2/s. Cerebrospinal fluid (CSF) analysis and cytology, and genetic analysis of CSF lymphoblasts confirmed a diagnosis of B-cell lymphoma. The owner opted for palliative treatment with prednisolone (1 mg/kg q12h); however, the cat died of dyspnoea 10 days after presentation. RELEVANCE AND NOVEL INFORMATION: CNS lymphomas, which are the second most common intracranial tumours in cats, are highly infiltrative lesions and radical surgical excision is not recommended. Therefore, accurate diagnosis is crucial. However, contrast-enhanced MRI cannot always differentiate these lesions from other conditions, including other CNS tumours and strokes. To the best of our knowledge, this is the first report to document the diffusion-weighted imaging features and apparent diffusion coefficient value for a feline CNS lymphoma. These findings are expected to improve the diagnostic accuracy of these lesions in cats.

Drip infusion cholangiography with CT in cats.

OBJECTIVES: Ultrasonography can detect extrahepatic biliary obstructions. However, visualisation of part of the bile duct using ultrasonography can be technically difficult if there is overlying bowel gas. This study investigated the safety and value of drip infusion cholangiography with computed tomography (DIC-CT) in the visualisation of the bile duct in healthy cats and extrahepatic biliary obstruction in cats with cholelithiasis. METHODS: DIC-CT was performed in three healthy cats and two cats with cholelithiasis. Meglumine iotroxate was administered by intravenous drip infusion over 30 mins. The attenuation value was measured perpendicular to the lumen by a region of interest covering about 50% of the area of the lumen. RESULTS: The bile ducts were visualised successfully with DIC-CT in all healthy cats without any significant adverse events. The attenuation value of the contrast medium was 271 ± 37 HU. In the two cats with cholelithiasis, the presence or absence of extrahepatic biliary obstruction was determined using DIC-CT without any significant adverse events. The attenuation value of cholelithiasis in the cat with an extrahepatic biliary obstruction was 933 ± 119 HU. In the cat with no extrahepatic biliary obstruction, the attenuation value of the contrast medium was 249 ± 53 HU and the attenuation value of cholelithiasis was 167 ± 28 HU. CONCLUSIONS AND RELEVANCE: DIC-CT visualises bile ducts and detects obstructive biliary disease without significant adverse events. Therefore, it appears to be a safe procedure for visualisation of the bile duct in cats.

Change in peripheral blood lymphocyte count in dogs following adoptive immunotherapy using lymphokine-activated T killer cells combined with palliative tumor resection.

We evaluated changes in peripheral blood lymphocyte (PBL) count in dogs following adoptive immunotherapy using lymphokine-activated T killer cells (T-LAK) in combination with surgery. Fifteen tumor-bearing dogs treated with T-LAK therapy combined with palliative resection of tumors were enrolled in the present study. T-LAK were generated from autologous peripheral blood mononuclear cells (PBMC) by culture with recombinant human interleukin -2 (rhIL-2) and solid phase anti-canine cluster of differentiation (CD)3 antibody. T-LAK were administrated intravenously at 2-4-week intervals. After the first administration of T-LAK, counts of PBL and T lymphocyte subsets (CD3(+), CD4(+) and CD8(+) cells) increased and the CD4/CD8 ratio decreased, with significant increases in CD8(+) cells (P<0.05). In 8 tumor-bearing dogs that were administered sequential T-LAK, available data on changes in PBL and T lymphocyte phenotypes until the fifth administration were also analyzed. In tumor-bearing dogs administered 5 rounds of T-LAK, CD8(+) cell counts were maintained high until the fifth administration of T-LAK. Moreover, the CD4/CD8 ratio remained low until the fifth administration of T-LAK. These results indicate that T-LAK therapy combined with surgery may increase peripheral blood T lymphocytes, particularly CD8(+) cells, in tumor-bearing dogs.

Elevation of serum surfactant protein-A with exacerbation in canine eosinophilic pneumonia.

A 7-year-old female spayed Labrador Retriever was admitted to our hospital, because of cough with sputum. She was diagnosed as having canine eosinophilic pneumonia (CEP) based on blood eosinophilia, bronchial pattern and infiltrative shadow observed on thoracic radiography, bronchiolar obstruction and air-space consolidation predominantly affecting the right caudal lung lobe, as revealed by computed tomography (CT), predominant eosinophils in CT-guided fine needle aspiration and the clinical course. She exhibited a good response to steroid therapy, and the cough disappeared. The serum surfactant protein (SP)-A level increased with the aggravated symptom and decreased markedly with improvement compared with the C-reactive protein level and the number of eosinophils. We propose that serum SP-A level is a good biomarker in CEP.

Computed tomography and magnetic resonance imaging of thoracic chordoma in a Bengal tiger (Panthera tigris tigris).

A Bengal tiger was presented for evaluation of weakness, ataxia and inappetance. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed a mass extending from the T7-8 vertebral body to the left rib and compressing the spinal cord. On CT, the bone destruction and sequestrum were shown. On MRI, the multilobulated mass appeared hypo- to isointense in T1-weighted and hyperintense in T2-weighted images. The tiger died after imaging, most likely from renal failure. Chordoma without metastasis was diagnosed on necropsy. The imaging characteristics were similar to those found in chordoma in humans. This report describes the use of CT and MRI in an exotic species.

Chordoma of the thoracic vertebrae in a Bengal tiger (Panthera tigris tigris).

A 19-year-old female Bengal tiger (Panthera tigris tigris) was presented with hind limb weakness, ataxia and respiratory distress. Computed tomography revealed a mass between the left side of the T7 vertebra and the base of the left 7th rib. The tiger then died, and necropsy was performed. Grossly, the vertebral mass was 6 × 5.7 × 3 cm, and invaded the adjacent vertebral bone and compressed the T7 spinal cord. Histologically, the mass was composed of large, clear, vacuolated and polygonal cells with osteochondral matrix. Cellular and nuclear atypia were moderate. The vacuolated cells stained positively for cytokeratin and vimentin and negatively for S-100. Based on these findings, the present case was diagnosed as a vertebral chordoma; the first report in a tiger.

Cloning and expression analysis of prohibitin mRNA in canine mammary tumors.

Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3'-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.

Treatment of a unicameral bone cyst in a dog using a customized titanium device.

A 4-year-old Shih-Tzu, referred for an enlarged left carpus, was diagnosed with a unicameral bone cyst. A customized titanium device was inserted into cystic lesion and fixed by titanium screws. Sufficient strength of the affected bone with the device inserted to maintain limb function was established after resection of contents of cystic lesion. There was no deterioration of the lesion of bone cyst, and acceptable function of the affected limb with no clinical signs of lameness was maintained during 36 months follow-up. The results of this study demonstrated that bone cyst curettage and use of a customized titanium device could provide an effective alternative treatment of huge lesion of unicameral bone cysts with the intent of preventing pathologic fractures.

In vivo evaluation of irinotecan-loaded QuadraSphere microspheres for use in chemoembolization of VX2 liver tumors.

PURPOSE: To investigate the pharmacokinetics and chemoembolization efficacy of irinotecan-loaded QuadraSphere microspheres (QSMs) in a rabbit VX2 liver tumor model. MATERIALS AND METHODS: Fourteen rabbits with VX2 liver tumors were divided into two groups. In the irinotecan-loaded QSM group (n = 7), 3 mg of QSMs (30-60 µm) containing 12 mg of irinotecan (0.6 mL; 20 mg/mL) were injected into the left hepatic artery. In the control group (hepatic arterial infusion [HAI] and QSMs; n = 7), 3 mg of QSMs suspended in ioxaglic acid were injected following a bolus injection of 0.6 mL of irinotecan solution (20 mg/mL). Sequential irinotecan, SN-38, and SN-38G concentration changes were measured in plasma within 24 hours and at 1 week and in tissues at 1 week. The VX2 tumor growth rates at 1 and 2 weeks were calculated from computed tomographic images. RESULTS: All rabbits underwent successful embolization. Plasma irinotecan, SN-38, and SN-38G concentrations in the irinotecan-loaded QSM group showed significantly sustained release compared with the control group (P = .01). Compared with the control group, the irinotecan-loaded QSM group had significantly higher irinotecan concentration in liver tumors (P = .03) and a tendency toward higher SN-38 concentration in liver tumors (P = .29). The SN-38G tissue concentrations were below the limits of quantification. The tumor growth rate was significantly lower and the tumor necrosis rate significantly higher in the irinotecan-loaded QSM group (P = .02 and P = .01, respectively). CONCLUSION: Chemoembolization via irinotecan-loaded QSMs more effectively suppresses tumor growth than chemoembolization with unloaded QSMs after HAI. A clinical feasibility study is warranted.

Relationship between major histocompatibility complex class I expression and prognosis in canine mammary gland tumors.

The aim of this study was to evaluate MHC class I expression and prognosis using tumor tissues surgically removed from 9 dogs with mammary gland carcinomas and from 13 dogs with complex carcinomas. We assessed MHC class I expression and its correlation with tumor size, B2M expression, infiltration of lymphocytes, histological grade and prognosis. Hematoxylin and eosin-stained sections were histologically graded using the Elston and Ellis grading method. MHC class I expression on tumor cells was evaluated using the avidin-biotin peroxidase complex method. Loss of MHC class I expression from canine mammary gland carcinomas was significantly correlated with poor prognosis (P<0.05). Loss of MHC class I expression showed no association with poor prognosis in canine mammary gland complex carcinomas, because the data were not balanced. Only 1 of 13 (7.6%) canine mammary gland complex carcinomas showed loss of MHC class I expression. All 13 of these dogs showed good prognosis. Thus, the low frequency of MHC class I expression loss from canine mammary gland complex carcinomas may be associated with good prognosis. Taken together, these results suggest that loss of MHC class I expression may be associated with poor prognosis in canine mammary gland carcinomas.

Surfactant protein-A concentration in sera from dogs with pulmonary parenchymal diseases.

Pulmonary surfactant protein A (SP-A) is used as a biomarker to understand the clinical features of pulmonary diseases and associated prognostic indices in human medicine. This study was conducted to investigate whether or not serum SP-A concentration can be used as a biomarker for identifying pulmonary parenchymal diseases in dogs. Thirty-two dogs with pulmonary parenchymal diseases, 34 with nonrespiratory diseases and 57 healthy dogs were included. Serum SP-A concentration was measured in all dogs using sandwich enzyme linked immunosorbent assay with an anti-dog SP-A polyclonal antibody. Median serum SP-A concentration in healthy dogs was <2.0 ng/ml, whereas that in dogs with aspiration pneumonia (n=11), primary lung tumors (n=9) and blunt traumatic lung injury (BTLI; n=12) was 3.1, 7.2 and 2.6 ng/ml, respectively; these values were significantly higher than those in healthy dogs. The serum SP-A concentration in dogs with nonrespiratory diseases was comparable with that in healthy dogs. No correlation was observed between the serum SP-A and plasma C-reactive protein concentrations in dogs with aspiration pneumonia and BTLI. There was a significant correlation between the serum SP-A concentration and thoracic radiographic changes in dogs with BTLI. These findings suggest that the serum SP-A concentration may be a useful clinical biomarker of alveolar damage that can be used for differential diagnosis of pulmonary parenchymal diseases and nonrespiratory diseases in dogs.

A spayed female cat with squamous cell carcinoma in the uterine remnant.

A 7-year-old spayed female domestic short-haired cat presented with dysuria and hematuria that had been unresponsive to medical therapy. Imaging tests such as ultrasonography, urethrocystography and computed tomography revealed a pelvic mass compressing the urethra. Based on histological examination of the mass following surgical resection, the cat was diagnosed squamous cell carcinoma (SCC) derived from the uterine remnant. After surgery, dysuria was resolved, but on instead, urine and fecal incontinence were observed. Then, about four months after surgery, recurrence of the mass and the symptoms was observed. Consequently, the cat was ultimately euthanized. This is the first report of SCC arising from the uterine remnant in a spayed female cat.

Incidental synovial myxoma with extensive intermuscular infiltration in a dog.

A 16-year-old male mixed-breed dog was euthanized due to progression of renal failure caused by renal adenocarcinoma in the left kidney. Apart from main symptomatic lesion, accumulation of transparent jelly-like fluid was observed between the right femoral muscles. Gross examination of the right hindlimb revealed multiple nodules in the articular surface and capsule of the stifle joints, which extended into the crural muscles. Histopathologically, the joint and intermuscular masses were characterized by variously-sized hypocellular nodules consisting of spindle to stellate cells suspended in an abundant myxoid matrix. There were cystic structures within the intermuscular masses, lined by synoviocyte-like cells. Based on the gross and histopathologic findings, the case was diagnosed as synovial myxoma with extensive intermuscular infiltration. Synovial myxoma should be considered in the differential diagnosis of dogs with myxomatous tumor between skeletal muscles, even in absence of joint or muscle symptoms.

Spinal cord trauma in a cat caused by ingestion of a splinter.

A 5-month-old male Abyssinian presented with right hemiplegia. Cervical radiographs, haematology and serum biochemistry were normal. A foreign body was identified on computed tomography (CT) and magnetic resonance imaging (MRI). Removal of the foreign body was carried out via a dorsal approach between the occipital bone and the C1 vertebral body using intra-operative CT. No complications were encountered. Thirty days after surgery, the cat was ambulatory.

Detection of chromogranin A in the adrenal gland extracts of different animal species by an enzyme-linked immunosorbent assay using Thomsen-Friedenreich antigen-specific Amaranthus caudatus lectin.

The reactivity of different lectins with crude chromogranin A (CgA) obtained from different animals, namely, cow, horse, dog, pig, and dolphin, was examined to identify lectin(s) that would be useful as coating reagent(s) in a sandwich enzyme-linked immunosorbent assay (ELISA). Of the different lectins studied, the Amaranthus caudatus lectin (ACA), which is specific for the Thomsen-Friedenreich (T)-antigen (Galß1-3GalNAc), was found to react with the CgA from different animals by western blotting. Purified rabbit anti-bovine CgA antibody was also found to cross-react with the crude CgA preparations. On the basis of these findings, a sandwich ELISA was developed with ACA as the coating reagent and anti-bovine CgA antibody as the probing antibody. Using this method, concentration-dependent curves ranging from 0.003 µg/mL to 25 µg/mL and from 0.02 µg/mL to 25 µg/mL were obtained for bovine CgA and canine CgA, respectively. Similarly, concentration-dependent curves were obtained for the equine, swine, and dolphin crude CgA extracts. Thus, ACA is concluded to be a valuable reagent for CgA detection in crude extracts from different animal species, and for CgA isolation/purification.

Acute myelomonocytic leukemia (AML-M4) in a dog with the extradural lesion.

A two-year-old dog having presented with neurological signs showed marked leukocytosis and appearance of blast cells in the peripheral blood. Hematological and bone marrow examination showed an increase in blasts having both myeloid and monocytic cells characteristics. The dog was diagnosed with acute myelomonocytic leukemia (AML-M4) on the basis of bone marrow findings. Although the dog was treated with a multi-combination chemotherapy, the neurological abnormalities progressed and the dog was euthanized. Myelographic examination and necropsy revealed the extradural lesion formed by AML-M4 around the cervical spinal cord and this lesion was considered as a cause of the neurological signs.

A rhabdomyosarcoma arising in the larynx of a dog.

A neoplastic nodular lesion, 2 × 3 cm in diameter, was found in the larynx of a 6-year-old spayed female dog. The tumor was ill-circumscribed, consisting histologically of large round cells with abundant cytoplasm interspersed with small round cells with less cytoplasm and occasional multinucleated cells (myotubes). Immunohistochemically, tumor cells were positive for myoglobin, desmin and vimentin in varying degrees, but negative for S-100 protein, GFAP or cytokeratin. Cytoplasmic myofilaments/myofibrils with a dense Z-line-like structure were seen, the fine structures of which were complemented by PTAH stain. Based on these findings, the tumor was diagnosed as a rhabdomyosarcoma, a very rare tumor in the larynx of dogs.

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors.

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors.

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.