Objective: To investigate effects of 1% hyaluronic acid–chondroitin sulfate–N-acetyl glucosamine (HCNAG) on the damage repair response in equine articular cartilage. Sample: Articular cartilage from 9 clinically normal adult horses. Procedures: Full-thickness cartilage disks were harvested from the third metacarpal bone. Cartilage was single-impact loaded (SIL) with 0.175 J at 0.7 m/s and cultured in DMEM plus 1 % (vol/vol) HCNAG or fibroblastic growth factor (FGF)-2 (50 ng/mL). Histologic and immunohistochemical techniques were used to identify tissue architecture and apoptotic cells and to immunolocalize type I and II collagen and proliferating nuclear cell antigen (PCNA). Results: Type II collagen immunoreactivity increased in SIL cartilage, compared with control samples. At days 14 and 28 (day 0 = initiation of culture), control samples had significantly fewer repair cells than did other treatment groups. In control samples and SIL + HCNAG, there was a significant decrease in apoptotic cell number, compared with results for SIL and SIL + FGF-2 samples. At days 14 and 28, there was a significant increase in chondrocytes stained positive for PCNA in the control samples. Conclusions and Clinical Relevance: 1% HCNAG significantly affected apoptotic and repair cell numbers in an SIL damage-repair technique in adult equine articular cartilage. However, HCNAG had no effect on the number of PCNA-positive chondrocytes or on type II collagen immunohistochemical results. The inclusion of 1% HCNAG in lavage solutions administered after arthroscopy may be beneficial to cartilage health by increasing the number of repair cells and decreasing the number of apoptotic cells.

OBJECTIVE To investigate protein kinase CK2 (CK2) expression in squamous cell carcinoma (SCC) of cats and to examine effects of CK2 downregulation on in vitro apoptosis and viability in SCC. SAMPLE Biopsy specimens of oral mucosa and testis and blood samples from clinically normal cats, biopsy specimens of oral SCC from cats, and feline SCC (SCCF1) and mammary gland carcinoma (K12) cell lines. PROCEDURES Immunohistochemically labeling for CK2α was performed on biopsy specimens. Sequences of the CK2α subunit gene and CK2α’ subunit gene in feline blood and feline cancer cell lines were determined by use of PCR and reverse-transcription PCR assays followed by direct Sanger sequencing. Specific small interfering RNAs (siRNAs) were developed for feline CK2α and CK2α'. The SCCF1 cells were treated with siRNA and assessed 72 hours later for CK2α and CK2α’ expression and markers of apoptosis (via western blot analysis) and for viability (via 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium assays). RESULTS CK2α was expressed in all feline oral mucosa samples and 7 of 8 oral SCC samples. Expression of CK2α and CK2α’ was successfully downregulated in SCCF1 cells by use of siRNAs, which resulted in decreased viability and induction of apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE In this study, CK2 appeared to be a promising therapeutic target for SCCs of cats. A possible treatment strategy for SCCs of cats would be RNA interference that targets CK2.

Objective-To determine cellular changes associated with secondary epidermal laminae (SEL) in forefeet and hind feet of ponies with insulin-induced laminitis. Animals-8 ponies. Procedures-Laminitis was induced in 4 ponies by IV administration of insulin and glucose; 4 control ponies received saline (0.9% NaCl) solution IV. Laminar tissue samples obtained from the dorsal aspects of the hooves were histologically evaluated. Primary epidermal lamina (PEL) length and width and SEL length, width, and angle were determined. Numbers of epidermal cell nuclei per micrometer and per total length of SEL and numbers of apoptotic and proliferative cells in axial, middle, and abaxial laminar regions were determined. Results-SEL in treatment group ponies were significantly longer, were significantly narrower, and had a smaller angle relative to PEL in all laminar regions versus control ponies. In treatment group ponies, the number of epidermal cell nuclei per SEL was typically higher and the number of cells per micrometer of SEL was lower in laminar regions, apoptotic cell numbers were higher in abaxial and middle regions in forefeet and hind feet, and proliferating cell numbers were higher in axial laminar regions in forefeet and all laminar regions in hind feet, versus control ponies. Conclusions and Clinical Relevance-Results indicated SEL elongation, narrowing, and alteration in orientation developed in all feet of ponies with insulin-induced laminitis. This was primarily attributable to cell stretching that developed at the same time as an accelerated cell death-proliferation cycle; differences in cell cycle responses among laminar regions between forefeet and hind feet may have been attributable to differences in load bearing.

Objective: To examine whether a zinc l-carnosine compound used for treatment of suspected gastric ulcers in dogs ameliorates acid-induced injury in canine gastric mucosa. Sample: Gastric mucosa from 6 healthy dogs. Procedures: Mucosa from the gastric antrum was harvested from 6 unadoptable shelter dogs immediately after euthanasia and mounted on Ussing chambers. The tissues were equilibrated for 30 minutes in neutral Ringer's solution prior to incubation with acidic Ringer's solution (HCl plus Ringer's solution [final pH, 1.5 to 2.5]), acidic Ringer's solution plus zinc l-carnosine compound, or zinc l-carnosine compound alone. Tissues were maintained for 180 minutes in Ussing chambers, during which permeability was assessed by measurement of transepithelial electrical resistance. After the 180-minute treatment period, tissues were removed from Ussing chambers and labeled with immunofluorescent anti–active caspase-3 antibody as an indicator of apoptosis. Results: Permeability of the gastric mucosa was significantly increased in a time-dependent manner by addition of HCl, whereas control tissues maintained viability for the study period. Change in permeability was detected within the first 15 minutes after acid application and progressed over the subsequent 150 minutes. The zinc l-carnosine compound had no significant effect on this increase in permeability. Apoptosis was evident in acid-treated tissues but not in control tissues. The zinc l-carnosine compound did not protect against development of apoptosis. Conclusions and Clinical Relevance: Addition of HCl caused a dose-dependent increase in gastric permeability over time and apparent induction of apoptosis as determined on the basis of immunofluorescence. However, there was no significant protective effect of a zinc l-carnosine compound. Nonetheless, results suggested the utility of this method for further studies of canine gastric injury.

Objective: To determine the effects of clenbuterol, at a dosage of up to 3.2 μg/kg for 14 days, PO, on skeletal and cardiac muscle in healthy horses undergoing treadmill exercise. Animals: 12 healthy horses from 3 to 10 years old. Procedures: Horses were randomly assigned to a control group (n = 6) or clenbuterol group (6) and received either saline (0.9% NaCl) solution or clenbuterol, PO, every 12 hours for 14 days. Horses were subjected to submaximal treadmill exercise daily during treatment. Muscle biopsy specimens were collected before and after treatment for determination of apoptosis. Echocardiographic measurements, serum clenbuterol and cardiac troponin I concentrations, and serum activities of creatine kinase and aspartate aminotransferase were measured before, during, and after treatment. Jugular venous blood samples were collected every 3 days during treatment. Echocardiography was repeated every 7 days after beginning treatment. Response variables were compared between treatment groups and across time periods. Results: No significant effect of clenbuterol or exercise on response variables was found between treatment and control groups at any time point or within groups over time. Conclusions and Clinical Relevance: Results did not reveal any adverse effects of treatment with an approved dose of clenbuterol on equine cardiac or skeletal muscle in the small number of horses tested.

Objective-To evaluate canine histiocytic sarcoma cell lines and tumor samples for dysregulation of the Kit/stem-cell factor (SCF), Flt3/Flt3 ligand (Flt3L), and Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling pathways, as these are known to contribute to the differentiation and survival of normal dendritic cells as well as malignant transformation of dendritic cells in mouse models. Sample Population-4 histiocytic sarcoma tumor cell lines and 35 formalin-fixed histiocytic sarcoma specimens obtained from dogs. Procedure-Histiocytic sarcoma cell lines were evaluated for expression of Kit/SCF, Flt3/Flt3L, and Met/HGF by use of reverse transcriptase-PCR procedures. Histiocytic sarcoma cell lines and tumor samples were evaluated for mutations in Kit, Flt3, and Met by use of PCR analysis of genomic DNA, followed by both sequencing and fluorescent PAGE for deletions or internal tandem duplications. The ability of the multitargeted split-kinase inhibitor SU11654 to block proliferation and induce apoptosis of histiocytic sarcoma cell lines was also evaluated. Results-No mutations in Kit, Flt3, and Met were identified in any of the cell lines or tumor samples evaluated. Furthermore, SU11654 did not induce cellcycle arrest or apoptosis of histiocytic sarcoma lines, even at supratherapeutic doses. Conclusions and Clinical Relevance-These data suggest that dysregulation of Kit/SCF, Flt3/Flt3L, and Met/HGF signaling pathways is unlikely to occur in histiocytic sarcomas of dogs and that inhibitors of the Kit, Flt3, and Met pathways are unlikely to provide clinical benefit to dogs with histiocytic sarcomas.