BACKGROUND: Open wounds affecting the distal part of limbs are commonly seen in horses. Due to certain factors, such as limited connective tissue available, potentiated growth of excessive granulation tissue, risk of contamination, and poor response to common treatments, healing of these wounds becomes a major problem for veterinarians on a number of occasions. Application of Mesenchymal Stem Cells (MSC) for enhancing wound healing has received a great deal of scientific attention. Among the MSCs, those derived from adipose tissue are frequently used owing to their availability, large number of cells after the primary harvest, and the capacity to differentiate to different cell lines.OBJECTIVES: The current study aimed to evaluate type 1 and 3 collagen genes expression in horse distal limb wounds treated via adipose-derived Mesenchymal Stem Cells and its comparison with bone marrow-derived stem cells.METHODS: After treatment of the experimental open wounds created in the distal limbs of four horses via autologous MSCs, real-time PCR was used for evaluating and comparing the expression of type I and III collagen genes in the healing wounds.RESULTS: Significant differences in the expression of type I and III collagen genes were observed between the treatment groups. Despite the fact that the greatest collagen genes expression belonged to bone marrow-derived MSCs, no significant differences were seen with adipose-derived MSCs.CONCLUSIONS: Owing to the advantages and an acceptable performance, adipose-derived MSCs could be considered as a novel approach to enhancing limb wound healing in horses.

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 3103 MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of ,0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and Veterinary medicine.

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 10(3) MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine.

Mesenchymal stem cells (MSCs) have ability to differentiate into multi-lineage cells, which confer a great promise for regenerative medicine to the cells. The aim of this study was to establish a method for isolation and characterization of adipose tissue-derived MSC (pAD-MSC) and bone marrow-derived MSC (pBM-MSC) in pigs. Isolated cells from all tissues were positive for CD29, CD44, CD90 and CD105, but negative for hematopoietic stem cell associated markers, CD45. In addition, the cells expressed the transcription factors, such as Oct4, Sox2, and Nanog by RT-PCR. pAD-MSC and pBM-MSC at early passage successfully differentiated into chondrocytes, osteocytes and adipocytes. Collectively, pig AD-MSC and BM-MSC with multipotency were optimized in our study.

Objective-To compare and correlate B-mode and color Doppler ultrasonographic characteristics with histopathologic findings of benign and malignant superficial lymph nodes in dogs. Study Population-50 superficial lymph nodes that were normal, abnormally large on physical examination, or represented regional lymph nodes draining an area of suspected primary malignancy in 30 dogs. Procedures-Before excision, lymph nodes were evaluated via B-mode and color Doppler ultrasonography to assess size, echogenicity, presence of a hilus, acoustic transmission, and vascular flow. Formalin-fixed, paraffin-embedded tissue sections of excised lymph nodes were stained with H&E and examined for the presence and extent of necrosis, fibrosis, fat, metastases, and tissue heterogeneity. To assess vascularity, the number and distribution of vessels stained by the Verhoeff van Gieson technique were recorded. Results-In superficial lymph nodes, a varied echogenicity corresponded to tissue heterogeneity. The ultrasonographic detection of a hilus was associated with the presence of fibrous tissue, fat, or both in the hilar region. Acoustic enhancement corresponded to presence of areas of intranodal necrosis. There was significant correlation between both the distribution and the number of vessels detected via ultrasonography and that detected by histopathology. The amount of flow estimated via ultrasonography was typically higher than that estimated via histologic examination. Conclusions and Clinical Relevance-Results indicated that histopathologic changes in canine lymph nodes have associated ultrasonographic changes and suggest that lymph node ultrasonography has an important role in the evaluation of lymph nodes in dogs in general and in dogs with neoplastic disease in particular.

OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44+, CD105+, and major histocompatability complex class II (MHCII; CD44+-CD105+-MHCII+ cells and CD44+-CD105+-MHCII− cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44+-CD105+-MHCII− CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.

Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia.

Objective: To isolate and characterize mesenchymal stem cells (MSCs) from canine muscle and periosteum and compare proliferative capacities of bone marrow-, adipose tissue-, muscle-, and periosteum-derived MSCs (BMSCs, AMSCs, MMSCs, and PMSCs, respectively). Sample: 7 canine cadavers. Procedures: MSCs were characterized on the basis of morphology, immunofluorescence of MSC-associated cell surface markers, and expression of pluripotency-associated transcription factors. Morphological and histochemical methods were used to evaluate differentiation of MSCs cultured in adipogenic, osteogenic, and chondrogenic media. Messenger ribonucleic acid expression of alkaline phosphatase, RUNX2, OSTERIX, and OSTEOPONTIN were evaluated as markers for osteogenic differentiation. Passage-1 MSCs were counted at 24, 48, 72, and 96 hours to determine tissue-specific MSC proliferative capacity. Mesenchymal stem cell yield per gram of tissue was calculated for confluent passage-1 MSCs. Results: Successful isolation of BMSCs, AMSCs, MMSCs, and PMSCs was determined on the basis of morphology; expression of CD44 and CD90; no expression of CD34 and CD45; mRNA expression of SOX2, OCT4, and NANOG; and adipogenic and osteogenic differentiation. Proliferative capacity was not significantly different among BMSCs, AMSCs, MMSCs, and PMSCs over a 4-day culture period. Periosteum provided a significantly higher MSC yield per gram of tissue once confluent in passage 1 (mean ± SD of 19,400,000 ± 12,800,000 of PMSCs/g of periosteum obtained in a mean ± SD of 13 ± 1.64 days). Conclusions and Clinical Relevance: Results indicated that canine muscle and periosteum may be sources of MSCs. Periosteum was a superior tissue source for MSC yield and may be useful in allogenic applications.

Nursing sickness, the largest single cause of mortality in adult female mink (Mustela vison), is an example of a metabolic disorder, which develops when the demands for lactation require extensive mobilization of body energy reserves. The condition is characterized by progressive weight loss, emaciation, and dehydration with high concentrations of glucose and insulin in the blood. Morbidity due to nursing sickness can be as high as 15% with mortality around 8%, but the incidence is known to vary from year to year. Stress has been shown to trigger the onset of the disease and old females and females with large litters are most often affected. Increasing demand for gluconeogenesis from amino acids due to heavy milk production may be a predisposing factor. Glucose metabolism is inextricably linked to that of protein and fats. In obesity (or lipodystrophy), the ability of adipose tissue to buffer the daily influx of nutrients is overwhelmed (or absent), interfering with insulin-mediated glucose disposal and leading to insulin resistance. Polyunsaturated fatty acids of the n-3 family play an important role in modulating insulin signalling and glucose uptake by peripheral tissue. The increasing demand on these fatty acids for milk fat synthesis towards late lactation may result in deficiency in the lactating female, thus impairing glucose disposal. It is suggested that the underlying cause of mink nursing sickness is the development of acquired insulin resistance with 3 contributing key elements: obesity (or lipodystrophy), n-3 fatty acid deficiency, and high protein oxidation rate. It is recommended that mink breeder females be kept in moderate body condition during fall and winter to avoid fattening or emaciation. A dietary n-3 fatty acid supplement during the lactation period may be beneficial for improved glycemic control. Lowering of dietary protein reduces (oxidative) stress and improves water balance in the nursing females and may, therefore, prevent the development and help in the management of nursing sickness. It is also surmised that other, thus far unexplained, metabolic disorders seen in male and female mink may be related to acquired insulin resistance.

OBJECTIVE To determine effects of hydrocortisone administration on serum leptin and adiponectin concentrations, abdominal fat distribution, and mRNA expression of leptin and adiponectin in abdominal adipose tissue of dogs. ANIMALS 12 healthy dogs. PROCEDURES Dogs received hydrocortisone (8.5 mg/kg; n = 6) or a placebo (6) orally every 12 hours for 90 days. Serum leptin and adiponectin concentrations were measured with a canine-specific ELISA on the day before (day 0; baseline) and during (days 1, 3, 7, 30, 60, and 90) administration. On days 0, 30, 60, and 90, abdominal fat mass was quantified with CT, and mRNA expression of leptin and adiponectin in abdominal fat was analyzed by use of a PCR assay. RESULTS Hydrocortisone administration resulted in an increase in visceral fat mass on days 60 and 90, compared with the mass at baseline. Visceral fat mass at the level of L3 increased during hydrocortisone administration. Serum leptin concentration began to increase on day 1 and was significantly higher than the baseline concentration on days 30 and 60. Serum adiponectin concentration on days 30, 60, and 90 was significantly lower than the baseline concentration. Leptin and adiponectin mRNA expression in abdominal fat was greater on day 30, compared with expression at baseline, but lower on days 60 and 90, compared with expression on day 30. Serum leptin concentration and visceral fat mass were correlated. CONCLUSIONS AND CLINICAL RELEVANCE Hydrocortisone administration affected abdominal fat distribution and serum leptin and adiponectin concentrations through dysregulation of leptin and adiponectin expression.