Enhanced chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells in zirconia microwell substrata.

In human cartilage tissue engineering, three-dimensional zirconia substrata have the potential advantage of producing many uniform cell clusters of controlled size without xenobiotic material, allowing easy clinical application. The objective of this study was to evaluate the possibility of using zirconia porous three-dimensional microwell substrata for chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells (BMMSCs) in vitro. In regular medium, 8 × 105, 2 × 106, and 5 × 106 equine BMMSCs from five thoroughbred horses were cultured on zirconia microwell substrata for 4 days to allow formation of clusters. The medium was replaced by chondrogenic culture medium. After chondrogenic culture for 7, 14 and 21 days, analysis of collagen type II alpha 1 gene (COL2A1) gene expression and observation of chondrogenic aggregates by scanning electron microscopy (SEM) were performed. SEM showed size-controlled cell clusters and increasing extracellular matrix over time when using 5 × 106 cells. The expression of COL2A1 on day 7 and 14 with 5 × 106 cells was significantly higher than that of conventional pellet culture with 2 × 106 cells. Histological evaluation by immunohistochemical staining for type II collagen (ColII) was performed after chondrogenic culture for 7 days. The clusters showed wide distribution of ColII. The results suggest that the zirconia substrata have the potential to enhance the chondrogenic differentiation of equine BMMSCs, allowing effective equine cartilage tissue engineering without xenobiotic materials.

Enteric peripheral neuroblastoma in a calf.

An 11-month-old female Japanese Black calf had showed chronic intestinal symptoms. A large mass surrounding the colon wall that was continuous with the colon submucosa was surgically removed. After recurrence and euthanasia, a large mass in the colon region and metastatic masses in the omentum, liver, and lung were revealed at necropsy. Pleomorphic small cells proliferated in the mass and muscular layer of the colon. The cells were positively stained with anti-doublecortin (DCX), PGP9.5, nestin, and neuron specific enolase (NSE). Thus, the diagnosis of peripheral neuroblastoma was made. This is the first report of enteric peripheral neuroblastoma in animals. Also, clear DCX staining signal suggested usefulness of DCX immunohistochemistry to differentiate the neuroblastoma from other small cell tumors in cattle.

Isolation of Aspergillus caninus (Synonym: Phialosimplex caninus) from a Canine Iliac Lymph node.

Aspergillus caninus (synonym: Phialosimplex caninus) is an anamorphic fungus species associated with systemic infections in dogs that has been transferred from the genus Phialosimplex to Aspergillus. Here, we report the first case of canine A. caninus infection in Japan. A castrated Japanese Shiba Inu (6 years old; weight, 12.5 kg) was referred to the Yamaguchi University Animal Medical Center, Yamaguchi, Japan, in June 2017 showing vitality loss and depression. Computed tomography revealed iliac and splenic hilum lymphopathies, and histologic examination of an iliac lymph node by biopsy revealed granulomatous lesions with numerous oval to round yeast-like fungal cells. Aspergillus caninus was isolated from the biopsy samples, and in vitro susceptibility tests of the isolate to the antifungal drugs amphotericin B (AMB), fluconazole (FLZ), itraconazole (ITZ), voriconazole (VRZ), and micafungin (MCF) were performed by the E-test method. The isolate from this dog exhibited a minimal inhibitory concentration of < 0.002 µg/ml to AMB, > 256 µg/ml to FLZ, < 0.002 µg/ml to ITZ, < 0.002 µg/ml to VRZ, and < 0.002 µg/ml to MCF, indicating that the isolate was not susceptible to FLZ and susceptible to AMB, ITZ, VRZ, and MCF. Since the response of the patient dog to ITZ and VRZ treatments was poor, more aggressive management using combination therapies of ITZ with other antifungals may be necessary for treating canine A. caninus infection in dogs.

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.

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.

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.