Objective-To determine effects of the glycoprotein IIb/IIIa receptor antagonists abciximab and eptifibatide on in vitro inhibition of cat platelets. Sample-Venous blood samples from 10 healthy cats. Procedures-Blood samples were anticoagulated with hirudin. Aliquots of whole blood from each cat were allocated to 5 treatments (baseline, 50 μg of abciximab/mL, abciximab volumetric control treatment, 4μM eptifibatide, and eptifibatide volumetric control treatment). Impedance platelet aggregometry was performed with 6.5μM ADP or 32μM thrombin receptor activator peptide (TRAP). Magnitude of platelet aggregation was determined by measuring the area under the curve 15 minutes after addition of ADP or TRAP. Results-Eptifibatide caused a significant reduction in platelet aggregation, compared with baseline values, for aggregometry with both ADP (median, 50.0; range, 8 to 122 [baseline median, 306.0; baseline range, 130 to 664]) and TRAP (median, 75.5; range, 3 to 148 [baseline median, 219.0; baseline range, 97 to 578]). There was no significant difference in platelet aggregation with abciximab, the abciximab volumetric control treatment, or the eptifibatide volumetric control treatment for aggregometry with ADP or TRAP. Conclusions and Clinical Relevance-Eptifibatide caused a significant reduction in platelet aggregation in vitro, but there was no identifiable antiplatelet effect for abciximab. Eptifibatide and abciximab have different binding and inhibitory actions; therefore, it can be hypothesized that abciximab would be ineffective in cats because of a lack of receptor binding, reduced binding kinetics, or lack of downstream signaling. Eptifibatide may be useful in identifying hyperreactive platelets in cats in an in vitro platelet inhibitory assay.

Objective—To evaluate the effects of clopidogrel on clinical and clinicopathologic variables in healthy horses with experimentally induced endotoxemia. Animals—12 adult mares. Procedures—Horses were assigned with a randomization procedure to receive clopidogrel (4 mg/kg, once, then 2 mg/kg, q 24 h; n = 6) or a placebo (6) through a nasogastric tube. After 72 hours of treatment, horses received lipopolysaccharide (LPS; 30 ng/kg, IV). Heart rate, respiratory rate, rectal temperature, CBC variables, plasma fibrinogen concentration, serum tumor necrosis factor-α concentration, plasma von Willebrand factor concentration, and measures of platelet activation (including ADP- and collagen-induced platelet aggregation and closure times, thrombelastography variables, and results of flow cytometric detection of platelet membrane P-selectin, phosphatidylserine, and microparticles) were determined at various times before and after LPS administration by investigators unaware of the treatment groups. Statistical analyses were performed with repeated-measures ANOVA. Results—4 of 6 clopidogrel-treated horses had significant decreases in ADP-induced platelet aggregation before and after LPS administration. Heart rate increased significantly after LPS administration only for the placebo group. No significant differences were detected between groups for CBC variables, closure time, and plasma concentration of fibrinogen or serum concentration of tumor necrosis factor-α, and no clinically relevant differences were detected for other hemostatic variables. Conclusions and Clinical Relevance—In this study, administration of LPS did not induce platelet hyperreactivity in horses on the basis of measures of platelet adhesion, aggregation, degranulation, and procoagulant activity. Administration of clopidogrel was associated with variable platelet antiaggregatory activity and attenuated some clinical signs of endotoxemia.

The objective of this study was to determine if prior measurement of the minimum alveolar concentration (MAC) of isoflurane influences the effect of ketamine on the MAC of isoflurane in dogs. Eight mixed-breed dogs were studied on 2 occasions. Anesthesia was induced and maintained using isoflurane. In group 1 the effect of ketamine on isoflurane MAC was determined after initially finding the baseline isoflurane MAC. In group 2, the effect of ketamine on isoflurane MAC was determined without previous measure of the baseline isoflurane MAC. In both groups, MAC was determined again 30 min after stopping the CRI of ketamine. Plasma ketamine concentrations were measured during MAC determinations. In group 1, baseline MAC (mean ± SD: 1.18 ± 0.14%) was decreased by ketamine (0.88 ± 0.14%; P < 0.05). The MAC after stopping ketamine was similar (1.09 ± 0.16%) to baseline MAC and higher than with ketamine (P < 0.05). In group 2, the MAC with ketamine (0.79 ± 0.11%) was also increased after stopping ketamine (1.10 ± 0.17%; P < 0.05). The MAC values with ketamine were different between groups (P < 0.05). Ketamine plasma concentrations were similar between groups during the events of MAC determination. The MAC of isoflurane during the CRI of ketamine yielded different results when methods of same day (group-1) versus separate days (group-2) are used, despite similar plasma ketamine concentrations with both methods. However, because the magnitude of this difference was less than 10%, either method of determining MAC is deemed acceptable for research purposes.

A high rate of mortality, expense, and complications of immunosuppressive therapy in dogs underscores the need for optimization of drug dosing. The purpose of this study was to determine, using a flow-cytometric assay, the 50% T-cell inhibitory concentration (IC50) of dexamethasone, cyclosporine, and the active metabolites of azathioprine (6-mercaptopurine) and leflunomide (A77 1726) in canine lymphocytes stimulated with concanavalin A (Con A). Whole blood was collected from 5 privately owned, healthy dogs of various ages, genders, and breeds. Peripheral blood mononuclear cells, obtained by density-gradient separation, were cultured for 72 h with Con A, a fluorochrome-tagged cell proliferation dye, and various concentrations of dexamethasone (0.1, 1, 10, 100, 1000, and 10 000 μM), cyclosporine (0.2, 2, 10, 20, 30, 40, 80, and 200 ng/mL), 6-mercaptopurine (0.5, 2.5, 50, 100, 250, and 500 μM), and A77 1726 (1, 5, 10, 25, 50, and 200 μM). After incubation, the lymphocytes were labeled with propidium iodide and an antibody against canine CD5, a pan T-cell surface marker. Flow cytometry determined the percentage of live, proliferating T-lymphocytes incubated with or without immunosuppressants. The mean (± standard error) IC50 was 3460 ± 1900 μM for dexamethasone, 15.8 ± 2.3 ng/mL for cyclosporine, 1.3 ± 0.4 μM for 6-mercaptopurine, and 55.6 ± 22.0 μM for A77 1722. Inhibition of T-cell proliferation by the 4 immunosuppressants was demonstrated in a concentration-dependent manner, with variability between the dogs. These results represent the initial steps to tailor this assay for individual immunosuppressant protocols for dogs with immune-mediated disease.

Objective—To determine the differentiation of canine adipose tissue–derived mesenchymal stem cells (ASCs) into endothelial progenitor cells (EPCs). Animals—3 healthy adult Beagles. Procedures—Canine ASCs were isolated and cultured from adipose tissue, and endothelial differentiation was induced by culturing ASCs in differentiation medium. Morphological and immunophenotypic changes were monitored. Expression of endothelial-specific markers was analyzed by conventional and real-time RT-PCR assay. The in vitro and in vivo functional characteristics of the endothelial-like cells induced from canine ASCs were evaluated by use of an in vitro solubilized basement membrane tube assay, low-density lipoprotein uptake assay, and in vivo solubilized basement membrane plug assay. Results—After differentiation culture, the cells developed morphological changes, expressed EPC markers such as CD34 and vascular endothelial growth factor receptor 2, and revealed functional characteristics in vitro. Furthermore, the induced cells allowed vessel formation in solubilized basement membrane plugs in vivo. Conclusion and Clinical Relevance—Results indicated that canine ASCs developed EPC characteristics when stimulated by differentiation medium with growth factors. Thus, differentiated canine ASC-EPCs may have the potential to provide vascularization for constructs used in regenerative medicine strategies.

Objective—To compare effectiveness and complications associated with peribulbar and retrobulbar anesthesia with bupivacaine in cats. Animals—6 healthy adult cats. Procedures—Cats were sedated with dexmedetomidine and received a peribulbar injection of 0.5% bupivacaine (1.5 mL), iopamidol (0.5 mL), and saline (0.9% NaCl) solution (1 mL) or retrobulbar injection of 0.5% bupivacaine (0.75 mL) and iopamidol (0.25 mL) in a crossover study with ≥ 2 weeks between treatments. The contralateral eye was the control. Injectate distribution was evaluated with CT. After atipamezole administration, periocular and corneal sensations, intraocular pressure (IOP), and ocular reflexes and appearance were evaluated for 24 hours. Results—All peribulbar and 3 of 6 retrobulbar injections resulted in CT evidence of intraconal injectate. Corneal sensation and periocular skin sensation were absent or significantly reduced relative to that for control eyes for 3 hours after peribulbar injection. Mean ± SD IOP immediately after injection was significantly higher for eyes with peribulbar injections (33 ± 12 mm Hg) than for control eyes or eyes with retrobulbar injections (both 14 ± 4 mm Hg) but 10 minutes later decreased to 18 ± 3 mm Hg. Exophthalmos, chemosis, and ptosis were evident in most injected eyes, and irritation was evident in 3 of 6 peribulbar-injected and 1 of 6 retrobulbar-injected eyes. All conditions resolved within 14 hours. Conclusions and Clinical Relevance—Peribulbar injection resulted in intraconal deposition of bupivicaine in a higher percentage of cats than did retrobulbar injection and induced notable anesthesia relative to that for the control eye; however, IOP increased temporarily.

Objective—To evaluate the ability of small interfering RNAs (siRNAs) to inhibit in vitro viral replication and gene expression of feline coronavirus (FCoV). Sample—Cell cultures of Crandell-Rees feline kidney cells. Procedures—5 synthetic siRNAs that each targeted a different region of the FCoV genome were tested individually and in various combinations for their antiviral effects against 2 strains of FCoV (feline infectious peritonitis virus WSU 79-1146 and feline enteric coronavirus WSU 79-1683) in cell cultures. Tested combinations targeted the FCoV leader and 3′ untranslated region, FCoV leader region and nucleocapsid gene, and FCoV leader region, 3′ untranslated region, and nucleocapsid gene. For each test condition, assessments included relative quantification of the inhibition of intracellular viral genomic RNA synthesis by means of real-time, reverse-transcription PCR analysis; flow cytometric evaluation of the reduction of viral protein expression in infected cells; and assessment of virus replication inhibition via titration of extracellular virus with a TCID50 infectivity assay. Results—The 5 siRNAs had variable inhibitory effects on FCoV when used singly. Combinations of siRNAs that targeted different regions of the viral genome resulted in more effective viral inhibition than did individual siRNAs that targeted a single gene. The tested siRNA combinations resulted in approximately 95% reduction in viral replication (based on virus titration results), compared with findings in negative control, nontargeting siRNA–treated, FCoV-infected cells. Conclusions and Clinical Relevance—In vitro replication of FCoV was specifically inhibited by siRNAs that targeted coding and noncoding regions of the viral genome, suggesting a potential therapeutic application of RNA interference in treatment of feline infectious peritonitis.

Objective—To characterize the response of skin of nonallergic horses following ID injection of polyclonal rabbit anti-canine IgE (anti-IgE) and rabbit IgG. Animals—6 healthy horses. Procedures—Skin in the cervical area was injected ID with anti-IgE and IgG. Wheal measurements and skin biopsy specimens were obtained before and 20 minutes and 6, 24, and 48 hours after injection. Tissue sections were evaluated for inflammatory cells at 4 dermal depths. Immunohistochemical analysis for CD3, CD4, and CD8 was performed, and cell counts were evaluated. Results—Anti-IgE wheals were significantly larger than IgG wheals at 20 minutes and 6 and 24 hours after injection. There were significantly more degranulated mast cells after anti-IgE injection than after IgG injection. There were significantly more eosinophils at 6, 24, and 48 hours and neutrophils at 6 hours after anti-IgE injection, compared with cell numbers at those same times after IgG injection. There were significantly more eosinophils in the deeper dermis of anti-IgE samples, compared with results for IgG samples. No significant differences between treatments were detected for CD3+, CD4+, or CD8+ cells. Conclusions and Clinical Relevance—Injection of anti-IgE antibodies was associated with the development of gross and microscopic inflammation characterized by mast cell degranulation and accumulation of inflammatory cells, particularly eosinophils and neutrophils. This pattern appeared to be similar to that of horses with naturally developing allergic skin disease, although lymphocytes were not increased; thus, ID injection of anti-IgE in horses may be of use for evaluating allergic skin diseases of horses.

Impaired abomasal motility is common in cattle with abomasal disorders. The macrolide erythromycin has been demonstrated to be an effective prokinetic agent in healthy calves and in adult cattle with abomasal volvulus or left displaced abomasum. We hypothesized that 2 structurally related macrolides, spiramycin and tulathromycin, would also be effective prokinetic agents in cattle. Six milk-fed, male, Holstein-Friesian calves were administered each of the following 4 treatments: spiramycin, 75 000 IU/kg BW, IM, this dose approximates 25 mg/kg BW, IM; tulathromycin, 2.5 mg/kg BW, SC; 2 mL of 0.9% NaCl (negative control); and erythromycin, 8.8 mg/kg BW, IM (positive control). Calves were fed 2 L of cow's milk containing acetaminophen (50 mg/kg body weight) 30 min after each treatment was administered and jugular venous blood samples were obtained periodically after the start of sucking. Abomasal emptying rate was assessed by the time to maximal plasma acetaminophen concentration. Spiramycin, tulathromycin, and the positive control erythromycin increased abomasal emptying rate compared to the negative control. We conclude that the labeled antimicrobial dose of spiramycin and tulathromycin increases the abomasal emptying rate in healthy milk-fed calves. Additional studies investigating whether spiramycin and tulathromycin exert a prokinetic effect in adult cattle with abomasal hypomotility appear indicated.

Objective-To evaluate the thermal antinociceptive and sedative effects and duration of action of tramadol hydrochloride after oral administration to American kestrels (Falco sparverius). Animals-12 healthy 3-year-old American kestrels. Procedures-Tramadol (5, 15, and 30 mg/kg) and a control suspension were administered orally in a masked randomized crossover experimental design. Foot withdrawal response to a thermal stimulus was determined 1 hour before (baseline) and 0.5, 1.5, 3, 6, and 9 hours after treatment. Agitation-sedation scores were determined 3 to 5 minutes before each thermal stimulus test. Results-The lowest dose of tramadol evaluated (5 mg/kg) significantly increased the thermal foot withdrawal thresholds for up to 1.5 hours after administration, compared with control treatment values, and for up to 9 hours after administration, compared with baseline values. Tramadol at doses of 15 and 30 mg/kg significantly increased thermal thresholds at 0.5 hours after administration, compared with control treatment values, and up to 3 hours after administration, compared with baseline values. No significant differences in agitation-sedation scores were detected between tramadol and control treatments. Conclusions and Clinical Relevance-Results indicated oral administration of 5 mg of tramadol/kg significantly increased thermal nociception thresholds for kestrels for 1.5 hours, compared with a control treatment, and 9 hours, compared with baseline values; higher doses resulted in less pronounced antinociceptive effects. Additional studies with other types of stimulation, formulations, dosages, routes of administration, and testing times would be needed to fully evaluate the analgesic and adverse effects of tramadol in kestrels and other avian species.