Objective-To assess the effects of ischemia and reperfusion on indicators of oxidative stress, activation of eosinophils, and apoptosis in the large colonic mucosa of horses. Animals-40 horses. Procedures-In 1 or two 20-cm-long segments of the pelvic flexure, ischemia was induced for 1 or 2 hours followed by no reperfusion or 30 minutes and 18 hours of reperfusion in anesthetized horses. Mucosal specimens were collected before (controls; n = 20 horses) and after each period of ischemia, and full-thickness tissue samples were collected after each period of reperfusion. Sections of colonic tissues were stained for histomorphometric analysis or assessment of eosinophil accumulation. Nitrotyrosine was identified immunohistochemically, and severity of apoptosis was determined via the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Results-Numbers of mucosal eosinophils were similar before induction of ischemia, after ischemia, and after ischemia-reperfusion. Eosinophil nitrotyrosine production increased significantly during ischemia and continued through 30 minutes of reperfusion; production was decreased at 18 hours of reperfusion but remained greater than that of the controls. In other leukocytes, nitrotyrosine generation peaked at 1 hour of ischemia and again at 18 hours of reperfusion. Compared with control findings, epithelial apoptosis increased gradually at 1 through 2 hours of ischemia with no further progression after reperfusion. Conclusions and Clinical Relevance-Results suggested that resident eosinophils in the large colon of horses react to mucosal injury from ischemia and reperfusion and may undergo oxidative stress under those conditions. Epithelial apoptosis could contribute to tissue damage.

Objective: To evaluate in vitro effects of gemcitabine alone and in combination with carboplatin on canine transitional cell carcinoma (TCC) cell lines. Sample: In vitro cultures of 5 canine TCC cell lines. Procedures: Cells were treated with gemcitabine, carboplatin, or a combination of both at various concentrations. Cell proliferation was assessed via a fluorescence-based microplate cell proliferation assay. Cell cycle was evaluated via propidium iodide staining, and apoptosis was assessed by measurement of caspase 3 and 7 enzymatic activity. Synergy between gemcitabine and carboplatin was quantified via combination index analyses. Results: Treatment of 5 canine TCC cell lines with gemcitabine or carboplatin decreased cell proliferation, increased apoptosis, and induced cell cycle arrest. Cell cycle arrest and apoptosis were markedly increased when cell lines were treated with both gemcitabine and carboplatin simultaneously or sequentially. Order of administration during sequential treatment did not consistently affect cell proliferation results in TCC cell lines. When TCC cell lines were treated with gemcitabine and carboplatin in combination at therapeutically relevant concentrations (gemcitabine concentration, < 10μM; carboplatin concentration, < 250μM), a significant decrease in cell proliferation was observed, compared with cell proliferation following treatment with gemcitabine or carboplatin alone. In combination, the effects of gemcitabine and carboplatin were synergistic in 3 of 5 cell lines and additive in the other 2. Conclusions and Clinical Relevance: Gemcitabine had antitumor effects on canine TCC cells in vitro, and the combination of gemcitabine and carboplatin had synergistic activity at biologically achievable concentrations.

Objective: To determine concentration-dependent effects of tiludronate on cartilage explants incubated with or without recombinant equine interleukin-1β (rEq IL-1). Sample: Articular cartilage explants from the femorotibial joints of 3 young adult horses. Procedures: Cartilage explants were incubated with 1 of 6 concentrations (0, 0.19, 1.9, 19, 190, or 1,900 mg/L) of tiludronate and with or without rEq IL-1 (0.01 ng/mL) for 96 hours. Prostaglandin E2 (PGE2) concentrations in culture medium and explant digests were analyzed via PGE2 enzyme immunoassay. Sulfated glycosaminoglycan (sGAG) concentrations in culture medium were quantified via 1,9-dimethylmethylene blue assay. Chondrocyte apoptosis in paraffin embedded explant sections was measured via terminal deoxynucleotidyl transferase-mediated dUTP nick end–labeling assay. Relative gene expression of matrix metalloproteinases (MMPs), interleukin (IL)-6, and IL-8 was determined via the comparative cycle threshold method. Results: rEq IL-1 increased PGE2 concentration, sGAG release from explants, chondrocyte apoptosis, and MMP gene expression. Lower tiludronate concentrations reduced rEq IL-1–induced sGAG release and chondrocyte apoptosis, whereas the higher tiludronate concentrations increased sGAG release and chondrocyte apoptosis. At the highest tiludronate concentration evaluated, IL-8 gene expression was increased independent of whether rEq IL-1 was present. Conclusions and Clinical Relevance: Tiludronate had biphasic concentration-dependent effects on cartilage explants that were independent of PGE2 secretion or MMP gene expression. Low tiludronate concentrations had some chondroprotective effects, whereas high tiludronate concentrations were detrimental to equine articular cartilage. Administration of tiludronate intra-articularly to horses may be detrimental, dependent on the dose used. In vivo studies are needed before intra-articular tiludronate administration to horses can be recommended.

Objective: To determine whether incubation of cruciate ligament cells with acetylsalicylic acid, carprofen, meloxicam, or robenacoxib provides protection against apoptosis induced by sodium nitroprusside (SNP). Sample: Explants of cranial (CCL) and caudal (CaCL) cruciate ligaments from eight 1-day-old Beagles. Procedures: Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of carprofen/mL; 0.1, 1, or 10 μg of meloxicam/mL; or 0.1, 1, or 10 μg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E2 concentrations were measured via an ELISA. Results: Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 μg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE2 production from SNP-damaged cells. Conclusions and Clinical Relevance: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments.

Classical swine fever (CSF) is a highly contagious disease among swine that has an important economic impact on worldwide. One clinical symptom of CSF is leukopenia, in particular lymphopenia, which is a characteristic event that occurs early in the course of CSF. Though lymphopenia associated with apoptosis, the pathogenic mechanism underlying the lymphopenia has not been well studied. To understand these mechanisms, we investigated the response of porcine B cell lines to infection with SW03, virulent strain isolated from swine tissue in Korea. This study demonstrated that SW03-infected L35 cell were induced apoptosis through the detection of activated caspase-3. In addition, SW03 infection leaded to alterations in pro-apoptotic, Bax, and anti-apoptotic, Bcl-xL proteins of Bcl-2 family. Our results would suggest that SW03-infected L35 cells induced apoptosis via intrinsic mitochondrial pathway.

Objective: To determine the oncolytic efficacy of an attenuated form of myxoma virus lacking the serp2 gene in canine tumor cells. Sample: Primary cells were isolated from tumors that were surgically removed from dogs and from connective tissue obtained from the cadaver of a dog. Cells of various established cell lines from tumors and nontumorous tissues were obtained. Procedures: Experiments were performed with cells in monolayer culture. Cell cultures were inoculated with wild-type myxoma viruses or myxoma viruses lacking the serp2 gene, and measures of cytopathic effects, viral growth kinetics, and cell death and apoptosis were determined. Results: Myxoma viruses replicated in cells of many of the primary and established canine tumor cell lines. Canine tumor cells in which expression of activated protein kinase B was upregulated were more permissive to myxoma virus infection than were cells in which expression of activated protein kinase B was not upregulated. Myxoma viruses lacking the serp2 gene caused more cytopathic effects in canine tumor cells because of apoptosis than did wild-type myxoma viruses. Conclusions and Clinical Relevance: Results of the present study indicated myxoma viruses lacking the serp2 gene may be useful for treatment of cancer in dogs. Impact for Human Medicine: Results of the present study may be useful for development of novel oncolytic treatments for tumors in humans.

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 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.