The objective of this study was to evaluate the ability of a long-term antioxidant-supplemented diet to regulate the oxidative stress and general health status of dogs involved in animal-assisted intervention (AAI) programs. Oxidative stress is a consequence of the accumulation of reactive oxygen species (ROS). Exercise-induced oxidative stress can increase muscle fatigue and fiber damage and eventually leads to impairment of the immune system. A randomized, placebo-controlled, crossover clinical evaluation was conducted with 11 healthy therapy dogs: 6 females and 5 males of different breeds and with a mean age of 2.7 ± 0.8 y (mean ± SEM). The dogs were divided into 2 groups, 1 fed a high quality commercial diet without antioxidants (CD) and the other a high quality commercial diet supplemented with antioxidants (SD) for 18 wk. After the first 18 wk, metabolic parameters, reactive oxygen metabolite-derivatives (d-ROMs), and biological antioxidant potential (BAP) levels were monitored and showed a significant reduction of d-ROMs, triglycerides, and creatinine values in the SD group (P < 0.05) and a significant increase in amylase values in the CD group (P < 0.01). At the end of this period, groups were crossed over and fed for another 18 wk. A significant decrease in amylase and glutamate pyruvate transaminase (GPT) values was observed in the CD and SD group, respectively (P < 0.05). In conclusion, a controlled, balanced antioxidant diet may be a valid approach to restoring good cell metabolism and neutralizing excess free radicals in therapy dogs.

Generation of free radicals and the ability of various antioxidants to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (ESR) techniques. Seven core cancellous bone specimens, 10 mm long and 7.9 mm in diameter, were obtained using aseptic technique, from the proximal portion of the humerus of 9 adult mixed-breed dogs. One core cancellous bone specimen from each dog was incubated in spin trap alpha-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium and served as a control. The other 6 specimens from each dog were incubated in alpha-phenyl-N-tert-butylnitrone/Eagle's minimum essential medium plus 1 of the following antioxidants: superoxide dismutase, catalase, superoxide dismutase/catalase, indomethacin, allopurinol, or deferoxamine mesylate. All specimens were incubated at 26 C for 90 minutes, then frozen at -20 C until they were prepared for analysis by ESR spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an ESR spectrum which was analyzed for hyperfine splitting constants to identify radicals. Each first-derivative spectrum was digitally double-integrated to obtain an area: these areas were used to compare intensities of the spin. For each treatment group, the areas from the treated specimens were compared with the areas from the control specimens, using a paired t-test. Significance was accepted at P less than or equal to 0.05. Spin adducts were detected in all cancellous bone specimens. Specimens incubated in deferoxamine (P = 0.0017) and superoxide dismutase/catalase (P = 0.0452) had significantly smaller areas than did control specimens. The areas for the other treatment groups did not differ significantly from controls. Our results substantiate free radical production in freshly procured cancellous bone specimens and that radical formation is attenuated by in vitro incubation with deferoxamine or superoxide dismutase/catalase.

Free, autogenous, full-thickness skin grafting was performed on 10 dogs; 5 dogs were given an iron chelator, deferoxamine-10% hydroxyethyl pentafraction starch (DEF-HES; 50 mg/kg of body weight, IV), and 5 dogs were given 10% hydroxyethyl pentafraction starch (HES) in 0.9% saline solution (5 ml/kg, IV). The percentage of viable graft on day 10 was higher, but not significantly so, in DEF-HES-treated dogs (mean +/- SD, 72.6 +/- 24.8%; median 76.5%) than in HES-treated dogs (mean +/- SD, 46.7 +/- 34.3%; median, 48.8%). A trend for a positive correlation between the percentage of viable graft (on day 10) and the percentage of original graft area (on day 28) was observed in HES- and DEF-HES-treated dogs; this trend was significant in HES-treated dogs (P = 0.012). Both groups had significant positive correlation between percentage of viable graft on day 10 and percentage of haired skin on day 28 (HES, P = 0.000002; DEF-HES, P = 0.0148). A unique finding in DEF-HES treated dogs was the consistent observation of foamy macrophages in the dermis adjacent to the grafts, in deep subcutaneous tissue below the grafts, and in normal dermis.

The scavenging of superoxide radicals by endogenous and therapeutically administered superoxide dismutases may prevent superoxide-mediated oxidative stress leading to lipid peroxidation, membrane lysis, and cell death in a wide variety of normal and pathologic states. Simple inorganic manganous salts such as MnCl2 also have superoxide dismutase-like activity and are extremely inexpensive, compared with enzymatic superoxide dismutase preparations. In this study, we explored the use of Mn salts as antioxidant drugs. We used the percentage of inhibition of nitroblue tetrazolium reduction by superoxide as a measure of the amount of superoxide dismutase-like activity. We found concentration-related increases in superoxide scavenging activity in simple buffer solutions upon addition of 1.25, 2.5, and 5.0 microM MnSO4. To determine whether Mn salts can inhibit oxidative damage in tissues, we used an in vitro model of lipid peroxidation in ischemic and reoxygenated rat liver slices. Concentrations of 10, 100, and 1000 micromoles MnCl2/L of buffer significantly decreased indicators of lipid peroxidation believed to be initiated by intracellular superoxide. We then determined the effectiveness of MnCl2 as a superoxide scavenger in conscious horses by measuring the superoxide scavenging ability of equine plasma before and during intravenous infusions of 1.0 L volumes of 0.9% saline solution containing 0, 12.5, or 25 mM MnCl2. Plasma Mn concentrations, which were determined by atomic absorption spectrophotometry, increased as a function of time and dose. Intravenously administered MnCl2 concomitantly produced dose-related increases in superoxide scavenging ability of equine plasma at 15, 30, 45, and 60 minutes after the onset of infusion, compared with preinfusion control values. Heart rate and blood pressure of the treated horses, which were monitored to measure toxicity of MnCl2, gradually increased in both treatment groups. Clinical adverse effects of MnCl2 administration included defecation, pawing, hyperexcitability, flank watching, and sweating. The results of this study indicate that simple Mn salts may scavenge superoxide radicals in vivo with minimal adverse reactions and at a trivial cost.

Sheets of mucosa from the jejunum of healthy horses were mounted in incubation chambers and bathed with Krebs-ringer bicarbonate solution. Changes in tissue function and histologic appearance were compared after the following conditions: (1) control conditions for 30 minutes with 95% O2/5% CO2 in the gas phase; (2) same conditions as control, except incubation with superoxide dismutase (300 U/ml) during the last 18 minutes; (3) anoxia for 15 minutes with 95% N2/5% CO2, followed by reoxygenation for 15 minutes; (4) same conditions as 3, except incubation with superoxide dismutase during reoxygenation; and (5) anoxia for 30 minutes. Anoxia reduced the accumulation of radiolabeled L-alanine and caused cell swelling, as indicated by an increase in tissue water and tissue Na contents. Reoxygenation improved the tissue's ability to accumulate L-alanine, but tissue swelling continued after this treatment. Tissue Na content and L-alanine accumulation were restored to control values by reoxygenation with superoxide dismutase in the bathing medium. The grade of structural damage, as indicated by separation of eptihelial cells from villi, was equally severe after all, but control, conditions. Superoxide dismutase had no effect on the tissue control conditions. Results of this study suggest that superoxide radicals are involved in the pathogenesis of reperfusion injury in equine jejunal mucosa and that this may be of clinical importance in cases of small intestinal strangulation obstruction.

Thirty horses were randomly assigned to 1 of 5 groups. All horses were anesthetized and subjected to ventral midline celiotomy, then the large colon was exteriorized and instrumented. Colonic arterial blood flow was reduced to 20% of baseline (BL) and was maintained for 3 hours. Colonic blood flow was then restored, and the colon was reperfused for an additional 3 hours. One of 5 drug solutions was administered via the jugular vein 30 minutes prior to colonic reperfusion: group 1, 0.9% NaCl; group 2, dimethyl sulfoxide: 1 g/kg of body weight; group 3, allopurinol: 25 mg/kg; group 4, 21-aminosteroid U-74389G: 10 mg/kg; and group 5, manganese chloride (MnCl2): 10 mg/kg. Hemodynamic variables were monitored and recorded at 30-minutes intervals. Systemic arterial, systemic venous (SV), and colonic venous (CV) blood samples were collected for measurement of blood gas tensions, oximetry, lactate concentration, PCV, and plasma total protein concentration. The eicosanoids, 6-keto prostaglandin F1alpha, prostaglandin E2, and thromboxane B2, were measured in CV blood, and endotoxin was measured in CV and SV blood. Full-thickness biopsy specimens were harvested from the left ventral colon for histologic evaluation and determination of wet weight-to-dry weight ratios (WW:DW). Data were analyzed, using two-way ANOVA for repeated measures, and statistical significance was set at P < 0.05. Heart rate, mean arterial pressure, and cardiac output increased with MnCl2 infusion; heart rate and cardiac output remained increased throughout the study, but mean arterial pressure returned to BL values within 30 minutes after completion of MnCl2 infusion. Other drug-induced changes were not significant. There were significant increases in mean pulmonary artery and mean right atrial pressures at 2 and 2.5 hours in horses of all groups, but other changes across time or differences among groups were not observed. Mean pulmonary artery pressure remained increased through 6 hours in all groups, but mean right atrial pressure had returned to BL values at 3 hours. Mean colonic arterial pressure was significantly decreased at 30 minutes of ischemia and remained decreased through 6 hours; however, by 3.25 hours it was significantly higher than the value at 3 hours of ischemia. Colonic arterial resistance decreased during ischemia and remained decreased throughout reperfusion in all groups; there were no differences among groups for colonic arterial resistance. Colonic venous PO2, oxygen content, and pH decreased, and PCO2 and lactate concentration increased during ischemia but returned to BL values during reperfusion. Compared with BL values, colonic oxygen extraction ratio was increased from 0.5 to 3 hours. By 15 minutes of reperfusion, colonic oxygen extraction ratio had decreased from the BL value in all groups and either remained decreased or returned to values not different from BL through 6 hours. Colonic venous 6-keto prostaglandin F1alpha and prostaglandin E2 concentrations increased during ischemia, but returned to BL on reperfusion; there were no changes in thromboxane2 concentration among or within groups. Endotoxin was not detected in CV or SV blood after ischemia or reperfusion. There were no differences among or within groups for these variables. Low-flow ischemia and reperfusion (I-R) of the large colon caused mucosal injury, as evidenced by increases in percentage of surface mucosal disruption, percentage depth of mucosal loss, mucosal hemorrhage, mucosal edema, mucosal interstitial-to-crypt ratio, mucosal neutrophil index, submucosal venular neutrophil numbers, and mucosal cellular debris index. There was a trend (P = 0.06) toward greater percentage depth of mucosal loss at 6 hours in horses treated with dimethyl sulfoxide, compared with the vehicle control solution. There were no differences in the remainder of the histologic variables among groups. Full-thickness and mucosal WW:DW increased with colonic I-R, but there were no differences among groups. There was a trend (P = 0.09) toward neutrophil accumulation, as measured by myeloperoxidase activity, in the lungs after colonic I-R, but there were no differences among groups. There was no change in lung WW:DW after colonic I-R. There were no beneficial effects of drugs directed against oxygen-derived free radical-mediated damage on colonic mucosal injury associated with low-flow I-R. Deleterious drug-induced hemodynamic effects were not observed in this study.