Objective: To compare the effects of autologous equine serum (AES) and autologous conditioned serum (ACS) on equine articular chondrocyte metabolism when stimulated with recombinant human (rh) interleukin (IL)-1β. Sample: Articular cartilage and nonconditioned and conditioned serum from 6 young adult horses. Procedures: Cartilage samples were digested, and chondrocytes were isolated and formed into pellets. Chondrocyte pellets were treated with each of the following: 10% AES, 10% AES and rhIL-1β, 20% AES and rhIL-1β, 10% ACS and rhIL-1β, and 20% ACS and rhIL-1β, and various effects of these treatments were measured. Results: Recombinant human IL-1β treatment led to a decrease in chondrocyte glycosaminoglycan synthesis and collagen II mRNA expression and an increase in medium matrix metalloproteinase-3 activity and cyclooxygenase-2 mRNA expression. When results of ACS and rhIL-1β treatment were compared with those of AES and rhIL-1β treatment, no difference was evident in glycosaminoglycan release, total glycosaminoglycan concentration, total DNA content, or matrix metalloproteinase-3 activity. A significant increase was found in chondrocyte glycosaminoglycan synthesis with 20% AES and rhIL-1β versus 10% ACS and rhIL-1β. The medium from ACS and rhIL-1β treatment had a higher concentration of IL-1β receptor antagonist, compared with medium from AES and rhIL-1β treatment. Treatment with 20% ACS and rhIL-1β resulted in a higher medium insulin-like growth factor-I concentration than did treatment with 10% AES and rhIL-1β. No difference in mRNA expression was found between ACS and rhIL-1β treatment and AES and rhIL-1β treatment. Conclusions and Clinical Relevance: Minimal beneficial effects of ACS treatment on proteoglycan matrix metabolism in equine chonrocytes were evident, compared with the effects of AES treatment.

Objective—To determine whether canine tumor cell lines express functional tissue factor and shed tissue factor-containing microparticles. Sample—Cell lines derived from tumors of the canine mammary gland (CMT12 and CMT25), pancreas (P404), lung (BACA), prostate gland (Ace-1), bone (HMPOS, D-17, and OS2.4), and soft tissue (A72); from normal canine renal epithelium (MDCK); and from a malignant human mammary tumor (MDA-MB-231). Procedures—Tissue factor mRNA and antigen expression were evaluated in cells by use of canine-specific primers in a reverse transcriptase PCR assay and a rabbit polyclonal anti-human tissue factor antibody in flow cytometric and immunofluorescent microscopic assays, respectively. Tissue factor procoagulant activity on cell surfaces, in whole cell lysates, and in microparticle pellets was measured by use of an activated factor X-dependent chromogenic assay. Results—Canine tissue factor mRNA was identified in all canine tumor cells. All canine tumor cells expressed intracellular tissue factor; however, the HMPOS and D-17 osteosarcoma cells lacked surface tissue factor expression and activity. The highest tissue factor expression and activity were observed in canine mammary tumor cells and pulmonary carcinoma cells (BACA). These 3 tumors also shed tissue factor-bearing microparticles into tissue culture supernatants. Conclusions and Clinical Relevance—Tissue factor was constitutively highly expressed in canine tumor cell lines, particularly those derived from epithelial tumors. Because tumor-associated tissue factor can promote tumor growth and metastasis in human patients, high tissue factor expression could affect the in vivo biological behavior of these tumors in dogs.

OBJECTIVE To evaluate the lipidomic profile of surfactant obtained from horses with asthma at various clinical stages and to compare results with findings for healthy horses exposed to the same conditions. SAMPLE Surfactant samples obtained from 6 horses with severe asthma and 7 healthy horses. PROCEDURES Clinical evaluation of horses and surfactant analysis were performed. Samples obtained from horses with severe asthma and healthy horses before (baseline), during, and after exposure to hay were analyzed. Crude surfactant pellets were dried prior to dissolution in a solution of isopropanol:methanol:chloroform (4:2:1) containing 7.5mM ammonium acetate. Shotgun lipidomics were performed by use of high-resolution data acquisition on an ion-trap mass spectrometer. Findings were analyzed by use of an ANOVA with a Tukey-Kramer post hoc test. RESULTS Results of lipidomic analysis were evaluated to detect significant differences between groups of horses and among exposure statuses within groups of horses. Significantly increased amounts of cyclic phosphatidic acid (cPA) and diacylglycerol (DAG) were detected in surfactant from severely asthmatic horses during exposure to hay, compared with baseline and postexposure concentrations. Concentrations of cPA and DAG did not change significantly in healthy horses regardless of exposure status. CONCLUSIONS AND CLINICAL RELEVANCE cPA 16:0 and DAG 36:2 were 2 novel lipid mediators identified in surfactant obtained from asthmatic horses with clinical disease. These molecules were likely biomarkers of sustained inflammation. Further studies are needed to evaluate a possible correlation with disease severity and potential alterations in the plasma lipidomic profile of horses with asthma.

Objective-To determine the effects of interleukin (IL)-6 and IL-1β stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. Sample-Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. Procedures-Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1β for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3β (GSK-3β), and β-catenin proteins in macroscopically normal cartilage samples and OCFs. Results-Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3β and coiled-coil domain containing 88C increased after 1 hour and expression of β-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5. Conclusion and Clinical Relevance-Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator β-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm2) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

Objective: To investigate the effects of gonadectomy on collagen homeostasis in cranial cruciate ligaments of male rabbits. Animals: 30 sexually immature (16-week-old) male New Zealand White rabbits. Procedures: Rabbits were randomly assigned to 5 groups of 6 rabbits each: sexually intact, placebo (control group); castrated, placebo; castrated, testosterone; castrated, dihydrotestosterone; and castrated, 17β-estradiol (E2). Control rabbits underwent a sham operation, and all other rabbits underwent gonadectomy. At the time of gonadectomy, the placebo and sex hormones were administered via slow-release pellets implanted subcutaneously as assigned. After 21 days of hormone supplementation, measurements were obtained of serum testosterone and E2 concentrations, ligament collagen characteristics, and androgen receptor, estrogen receoptor α, and matrix metalloproteinase expression. Results: Following gonadectomy and hormone supplementation, the treatment groups differed in serum testosterone and E2 concentrations to various degrees. Collagen concentrations were lower and fiber diameters higher in the absence of sex hormones, in association with the degrees of estrogen receptor a and androgen receptor expression. Although differences were detected among the groups in matrix metalloproteinase expression, these differences were not significant. Conclusions and Clinical Relevance: Sex hormones appeared to play a role in cranial cruciate ligament homeostasis in male rabbits. Physiologic changes triggered by the lack of sex hormones following gonadectomy in sexually immature rabbits may potentially predispose those rabbits to orthopedic injuries.

The development and availability of follicles is an indicator to predict which of the follicle sizes are suitable to recover the oocytes assisted by means of ultrasonography of transvaginal oocyte retrieval (OPU). Thus, the study was done in order to characterize the follicular recruitment and distribution in response to the repeated removal of follicles, and thus to determine the availability of follicles and immature oocytes harvested repeatedlyfor two consecutive days of OPU in beef cows. Results indicated that 24-h OPU showed significantly greater numbers of medium and large follicles than small categories (P<0.05). However the 48-hr of OPU does not showed any differences of follicles categories (P>0.05). The mean total number of follicles and immature oocytes recovered were higher (P<0.05) in 24-hr OPU (13.76±1.2 and 7.38 ± 1.7) compared to 48-hr OPU (9.08 ± 1.5 and 3.54 ± 1.00) with the oocyte retrieval rate of 51.22% and 38.17%, respectively. The morphological classification indicated the 24-hr oocyte retrieval produced 62% of suitable immature oocytes that can be used for in vitro embryo production. In conclusion, the repeated removal of two consecutive days of OPU has averted the development of dominant follicle, and thus, gave an atmosphere to the subordinate follicles to continue growth relatively to an equal proportion of small, medium and large categories of follicles. Due to the reduction of follicle and recovery rate at 48-hr it is suggested that OPU be carried out later than 48 hour so that the follicle has more time to increase the diameter size.

A number of studies have measured collagen fibers and collagen&#xD;deposition in wound healing process with advances imaging techniques. However, these are performed by complicated methods and need specific tools. In search of the easier ways in routine histopathological laboratory, collagen measurement and staining pattern of wound healing process were observed in wounded skin of Sprague Dawley’s rat by using two different stains which are standard haematoxylin and eosin (H&E) and modified Masson’s&#xD;trichrome staining (MT). The comparison between these staining in wounded tissues was made to evaluate the advantages and disadvantages of both staining in wound healing study for 21 days postwounding. Tissues which stained with MT staining was then evaluated its collagen re-organization and density by using polarized light microscope with the aid of image analyzer software. Results showed that tissues stained with standard H&E could not be used to measure and differentiate the collagen deposition which is contradictory to MT staining. Wounded tissue stained with MT staining has showed a clear view of collagen fibers deposition&#xD;and re-organisation compared to H&E staining. This finding could validate the using of modified MT staining which leads to accurate histopathological analysis and observation in wound healing study.