Novel Approach for Evaluating Pregnancy-Associated Glycoprotein and Inflammation Markers during the Postpartum Period in Holstein Friesian Cows.

We evaluated the relationship between decreased pregnancy-associated glycoprotein (PAG) levels, inflammatory parameters (serum amyloid A [SAA] and milk amyloid A [MAA]), postpartum inflammatory conditions (mastitis, ketosis, and follicular cysts), and the FOXP3 gene. Nineteen Holstein-Friesian cows were included in this study. Up to approximately eight weeks after delivery, weekly health examinations were performed for mastitis and ketosis, and reproductive organ ultrasonography was performed. The decreasing PAG rate was negatively correlated with SAA concentration (r = -0.493, p = 0.032). Cows with mastitis exhibited a slower trend of PAG decrease (p = 0.095), and a greater percentage of these cows had MAA concentrations above 12 µg/mL (p = 0.074) compared with those without mastitis. A negative correlation, although nonsignificant (r = -0.263, p = 0.385), was observed between the day-open period and decreased PAG rate. The day-open period was correlated with the presence or absence of follicular cysts (p = 0.046). Four cows that developed follicular cysts were homozygous for the G allele of the FOXP3 gene related to repeat breeders. These results indicate a relationship between a decreased PAG rate and inflammatory status during the postpartum period. Thus, suppressing inflammation during the perinatal period may improve reproductive efficiency in the dairy industry.

Relationship Between Ovary Size and Anti-Müllerian Hormone Levels in Holstein-Friesian Cows.

The aim of this study was to verify the association between ovarian size and blood AMH levels in HF cows. Sixty multiparous HF cows from three herds were included in this study. The data required for calculating the ovarian volume included the "major axis (length)," "minor axis (width)," and "thickness" of the ovary. All ultrasonography (US) images were acquired at the outermost ends/poles of both the ovaries and of the follicles (>8 mm) and corpus luteum (CL); concomitantly, the blood was sampled from the jugular or coccygeal vein. Based on the ovarian images of each cow, the following ovarian size patterns were calculated using an image analysis software: (1) total area of both the left and right ovaries, (2) individual size of the large ovary, and (3) individual size of the small ovary. For each ovary area pattern, two properties were assessed: (A) presence of follicles (>8 mm) and CL, which may not secret AMH, in the ovaries and (B) absence of follicles (>8 mm) and CL in the ovaries. Serum AMH levels were measured using enzyme-linked immunosorbent assay. The correlation between ovary size and serum AMH levels was measured in terms of the aforementioned patterns and was evaluated statistically. The results of our preliminary study with ovaries from slaughter-house cows (n = 22) revealed that the "thickness" of the ovary was not necessary for estimating ovarian volume and that length and width were sufficient. A strong correlation was observed among ovarian length, width, and thickness (r > 0.96). No significant difference was observed (p > 0.05) in the mean ages or parities among the three herds. Among the ovary sizes measured in this study, the highest correlation was found between the total size of an individual large ovary (including follicular and luteal size) and AMH levels (r = 0.387, p = 0.002). This is the first study to demonstrate the correlation between total size of individual large ovaries and serum AMH levels in HF cows. US observations of the ovaries will allow for estimation of differences in AMH levels and help predict ovarian activity and superovulation performance of cows.

Epidemiological survey, general blood biochemistry, and histological examination of slaughtered heavy horse breeds with hemorrhage in the adipose tissue in the crest of the neck.

Fifty-four slaughtered horses were classified into groups having adipose tissue in the crest of the neck with or without hemorrhage (AH and NH groups, respectively). Blood biochemical tests (Alb, TP, T-bil, GOT, GPT, LDH, T-cho, and BUN) and an epidemiological survey (age, gender, weight, origin, breed, BCS, CNS, and hoof disease) were performed. T-bil tended to be high, while the other parameters were normal. Weight, BCS, and CNS were higher in the AH group (P<0.05). GOT was lower in the AH group (P<0.05). It was suspected that the horses in the AH group had lipomatosis. It was assumed that the adipose tissue of the horses in the AH group contained damaged capillaries, and inflammation was confirmed based on evidence of macrophages and lymphocytes.

Effect of a novel rice fermented extract on gastric ulcers in horses.

Gastric ulcers cause appetite loss, poor body condition, and colic in horses. This study investigated the protective effect of a rice fermented extract on the gastric mucosa in 17 healthy Thoroughbreds. For one month, horses in the rice fermented extract (nine horses) and control (eight horses) groups were orally administered a rice fermented extract (100%; 0.2 ml/kg, SID) and tap water (0.2 ml/kg), respectively. Gastric endoscopic images were obtained before and one month after rice fermented extract administration. The gastric ulcer score was lower after administration (median, 1; maximum, 2; minimum, 1) than before administration (median, 4; maximum, 4; minimum, 3) in the rice fermented extract group (P<0.05). In conclusion, the administration of a rice fermented extract for one month improves gastric mucosal lesions in Thoroughbreds with gastric ulcers.

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.

Liver regeneration therapy through the hepatic artery-infusion of cultured bone marrow cells in a canine liver fibrosis model.

BACKGROUND: We previously reported regenerative therapies for decompensated cirrhosis based on peripheral venous drip infusion using non-cultured whole bone marrow (BM) cells, or the less invasive cultured BM-derived mesenchymal stem cells (BMSCs). Here, we assessed the efficacy and safety of hepatic arterial infusion using cultured autologous BMSCs, comparing it with peripheral infusion, using our established canine liver fibrosis model. METHODS: Canine BM cells were harvested and cultured, and the resultant BMSCs were returned to carbon tetrachloride (CCl4)-induced liver cirrhosis model canines via either a peripheral vein (Vein group) or hepatic artery (Artery group). A variety of assays were performed before and 4, 8, and 12 weeks after BMSC infusion, and liver fibrosis and indocyanine green (ICG) half-life (t1/2) were compared to those in a control group that received CCl4 but not BMSCs. The safety of this approach was evaluated by contrast-enhanced computed tomography (CT) and serial blood examinations after infusion. RESULTS: Four weeks after infusing BMSCs, a significant improvement was observed in the Vein group (n = 8) compared to outcome in the Control group (n = 10), along with a decrease in ICG t1/2. In the Artery group (n = 4), ICG t1/2 was significantly shorter than that in the Vein group at 8 weeks (Δt1/2: -3.8 ± 1.7 min vs. +0.4 ± 2.4 min; p < 0.01) and 12 weeks (Δt1/2: -4.2 ± 1.7 min vs. +0.4 ± 2.7 min; p < 0.01) after BMSC administration. Post-infusion contrast-enhanced CT showed no liver infarction, and blood tests showed no elevations in either serum lactate dehydrogenase concentrations or hypercoagulability. CONCLUSIONS: We confirmed the efficacy and safety of the hepatic arterial infusion of cultured autologous BMSCs using a canine model, thereby providing non-clinical proof-of-concept.

Canine Liver Fibrosis Model to Assess the Functions of Infused Autologous Bone Marrow-Derived Cells.

Liver cirrhosis is the end stage of chronic liver disease, and the only radical treatment for decompensated liver cirrhosis is still liver transplantation. Development of effective regenerative therapy for liver cirrhosis is an urgent task. Before human clinical trials can be considered, the safety and efficacy of any planned protocol must be confirmed in medium-to-large animals. Therefore, we have developed a novel canine liver fibrosis model for proof of concept (POC) studies.

Agenesis of hepatic lobes in a dog.

Agenesis of a hepatic lobe is an extremely rare congenital anomaly and only one dog have been reported in veterinary literature. We encountered a dog with this anomaly diagnosed by Computed tomography (CT) and portography. A two-year-old, 6.9-kg female Shih tzu dog was presented with vomiting and anorexia. The dog had no history of abdominal surgery or trauma. Biochemical analysis showed elevated plasmatic liver enzymes. CT revealed the absence of the liver parenchyma and vascular system of the left lobe, quadrate lobe and papillary process of the caudate lobe. A portosystemic shunt was also observed. The liver parenchyma and vascular system of these lobes were not detected under digital subtraction angiography during laparotomy. Furthermore, the liver parenchyma and vascular system of these lobes were not detected even when the remaining liver volume increased two months after treating the shunt vessel. CT proved itself a good option for antemortally diagnosis of hepatic agenesis in a dog.

Aldehyde dehydrogenase activity helps identify a subpopulation of murine adipose-derived stem cells with enhanced adipogenic and osteogenic differentiation potential.

AIM: To identify and characterize functionally distinct subpopulation of adipose-derived stem cells (ADSCs). METHODS: ADSCs cultured from mouse subcutaneous adipose tissue were sorted fluorescence-activated cell sorter based on aldehyde dehydrogenase (ALDH) activity, a widely used stem cell marker. Differentiation potentials were analyzed by utilizing immunocytofluorescece and its quantitative analysis. RESULTS: Approximately 15% of bulk ADSCs showed high ALDH activity in flow cytometric analysis. Although significant difference was not seen in proliferation capacity, the adipogenic and osteogenic differentiation capacity was higher in ALDHHi subpopulations than in ALDHLo. Gene set enrichment analysis revealed that ribosome-related gene sets were enriched in the ALDHHi subpopulation. CONCLUSION: High ALDH activity is a useful marker for identifying functionally different subpopulations in murine ADSCs. Additionally, we suggested the importance of ribosome for differentiation of ADSCs by gene set enrichment analysis.

Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells.

This study aimed to demonstrate single-cell phosphospecific flow cytometric analysis of canine and murine adipose-derived stem/stromal cells (ADSCs). ADSCs were obtained from clinically healthy laboratory beagles and C57BL/6 mice. Cell differentiation into adipocytes, osteocytes, and chondrocytes was observed for the cultured canine ADSCs (cADSCs) and murine ADSCs (mADSCs) to determine their multipotency. We also performed single-cell phosphospecific flow cytometric analysis related to cell differentiation and stemness. Cultured cADSCs and mADSCs exhibited the potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, single-cell phosphospecific flow cytometric analysis revealed similar ß-catenin and Akt phosphorylation between mADSCs and cADSCs. On the other hand, it showed the phosphorylation of different Stat proteins. It was determined that cADSCs and mADSCs show the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Furthermore, a difference in protein phosphorylation between undifferentiated cADSCs and mADSCs was identified.

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.

Aldehyde dehydrogenase activity identifies a subpopulation of canine adipose-derived stem cells with higher differentiation potential.

Adipose-derived stem cells (ADSCs) are abundant and readily obtained, and have been studied for their clinical applicability in regenerative medicine. Some surface antigens have been identified as markers of different ADSC subpopulations in mice and humans. However, it is unclear whether functionally distinct subpopulations exist in dogs. To address this issue, we evaluated aldehyde dehydrogenase (ALDH) activity-a widely used stem cell marker in mice and humans-by flow cytometry. Approximately 20% of bulk ADSCs showed high ALDH activity. Compared to cells with low activity (ALDHLo), the high-activity (ALDHHi) subpopulation exhibited a higher capacity for adipogenic and osteogenic differentiation. This is the first report of distinct ADSC subpopulations in dogs that differ in terms of adipogenic and osteogenic differentiation potential.

Craniocervical junction abnormalities with atlantoaxial subluxation caused by ventral subluxation of C2 in a dog.

Craniocervical junction abnormalities with atlantoaxial subluxation caused by ventral subluxation of C2 were diagnosed in a 6-month-old female Pomeranian with tetraplegia as a clinical sign. Lateral survey radiography of the neck with flexion revealed atlantoaxial subluxation with ventral subluxation of C2. Computed tomography revealed absence of dens and atlanto-occipital overlapping. Magnetic resonance imaging showed compression of the spinal cord and indentation of caudal cerebellum. The diagnosis was Chiari-like malformation, atlantoaxial subluxation with ventral displacement of C2, atlanto-occipital overlapping, and syringomyelia. The dog underwent foramen magnum decompression, dorsal laminectomy of C1, and ventral fixation of the atlantoaxial joint. Soon after the operation, voluntary movements of the legs were recovered. Finally, the dog could stand and walk without assistance. The dog had complicated malformations at the craniocervical junction but foramen magnum decompression and dorsal laminectomy for Chiari-like malformation, and ventral fixation for atlantoaxial subluxation resulted in an excellent clinical outcome.

Transcatheter arterial embolization in normal canine liver.

OBJECTIVE: To determine the effects of selective transcatheter arterial embolization (TAE) in the normal canine liver. STUDY DESIGN: Experimental study. ANIMALS: Adult Beagle dogs (n = 5). METHODS: Gelatin sponge particles (GSPs) were injected through a microcatheter for selective embolization of the left hepatic artery in normal dogs. Computed tomography (CT) and histology were performed during an 8-week observation period; biochemical analysis data were obtained during a 12-week observation period after TAE. RESULTS: Embolization was successful in all dogs and did not induce any change in the clinical appearance of dogs. Postoperative CT was consistent with recanalization of the artery within 2 weeks of embolization in all dogs. Hepatic enzyme levels increased temporarily after embolization but gradually returned to normal ranges. Histological examinations did not differ between treated and untreated liver tissues. CONCLUSION: TAE appears safe in normal dogs observed for 12 weeks. Arterial recanalization seems to occur within 2 weeks after injection of GSPs in the left hepatic artery. IMPACT/CLINICAL RELEVANCE: Selective TAE of the hepatic artery was well tolerated in normal dogs. Selective TAE may be applicable to canine hepatocellular carcinoma.

A canine liver fibrosis model to develop a therapy for liver cirrhosis using cultured bone marrow-derived cells.

We have been developing a therapy for liver cirrhosis using cultured autologous bone marrow-derived mesenchymal stem cells (BMSCs). Before human clinical trials can be considered, the safety and efficacy of BMSC infusion in medium to large animals must be confirmed; thus, we developed a canine liver fibrosis model. A small amount of bone marrow fluid was aspirated from the canine humerus to assess the characteristics of BMSCs. We implanted a venous catheter in the stomach and a subcutaneous infusion port in the back of the neck of each canine. Repeated injection of CCl4 through the catheter was performed to induce liver cirrhosis. After 10 weeks of CCl4 injection, eight canines were equally divided into two groups: no cell infusion (control group) and autologous BMSC infusion through the peripheral vein (BMSC group). A variety of assays were carried out before and 4 weeks after the infusion. The area of liver fibrosis stained with sirius red was significantly reduced in the BMSC group 4 weeks after BMSC infusion, consistent with a significantly shortened half-life of indocyanine green and improved liver function. Conclusion: We established a useful canine liver fibrosis model and confirmed that cultured autologous BMSC infusion improved liver fibrosis without adverse effects. (Hepatology Communications 2017;1:691-703).

Anatomical evaluation of hepatic vascular system in healthy beagles using X-ray contrast computed tomography.

Liver contrast X-ray computed tomography (CT) has been used for evaluation of hepatic vessels for liver transplantation, liver lobectomy, interventional radiology and diagnosis of hepatocellular carcinoma in humans. However, there remains scant available anatomical information on normal hepatic vessels in the veterinary field. In this study, visualization of hepatic vessels was evaluated in 32 normal beagle dogs by X-ray contrast CT using triple phase images. The following hepatic vessels were clearly visualized: arterial, portal and hepatic veins. With regards to the running patterns of the portal vein and hepatic vein, there were no significant differences between the dogs. However, the hepatic artery exhibited some differences in each dog. In particular, the hepatic artery of the quadrate lobe and the right lateral lobe had many running patterns. The results of the present study could be useful for veterinary diagnosis, surgery and interventional radiology.

High temperature hyperthermia treatment for canines exhibiting superficial tumors: A report of three cases.

High temperature hyperthermia (HTH) treatment has previously been demonstrated to suppress tumor growth in a tumor-bearing rat model. In the present study, the effects of HTH therapy for the treatment of spontaneous tumors in canines was evaluated. In case 1, an 18-year-old female Papillon presented with a right forelimb rhabdomyosarcoma. Case 2 was a 13-year-old male English Cocker Spaniel with a right external auditory canal ceruminous adenocarcinoma and case 3 was a 14-year-old male Golden Retriever that exhibited a perianal gland adenocarcinoma, which surrounded the anus. HTH treatment was performed in all three cases for 10 min at 45-65°C with or without the inhalation of isoflurane. In case 1, the tumor disappeared four weeks following HTH treatment. In case 2, the tumor volume had decreased by day 21, and in case 3, HTH was performed three times and the tumor disappeared following the third procedure. HTH is considered to be a simple procedure with no severe side effects. Consequently, this treatment modality is hypothesized to become a useful alternative therapy for superficial tumors in companion animals.

Canine bone marrow stromal cells promote functional recovery in mice with spinal cord injury.

Regenerative therapy has begun to be clinically applied in humans and dogs to treat neurological disorders, such as spinal cord injury (SCI). Here, we show the therapeutic potential of transplantation of cultured canine bone marrow stromal cells (BMSCs) into mice with SCI. Canine BMSC transplantation therapy was performed, immediately after the spinal cord was injured. Canine BMSC therapy enhanced functional recovery of the hind limbs in mice with SCI. Nestin-positive cells were observed only in the lesion of mice with SCI that received BMSCs. These results suggest that canine BMSCs promote functional recovery in mice with SCI and that migration of nestin-positive cells may contribute to the efficacy of the BMSC treatment.