OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44+, CD105+, and major histocompatability complex class II (MHCII; CD44+-CD105+-MHCII+ cells and CD44+-CD105+-MHCII− cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44+-CD105+-MHCII− CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.

The aim of this study was to determine the efficacy and pharmacokinetics of bupivacaine in combination with epinephrine or dexmedetomidine after intraperitoneal administration in cats undergoing ovariohysterectomy. Sixteen healthy adult cats (3.3 ± 0.6 kg) were included in a prospective, randomized, masked clinical trial after obtaining owners' consent. Anesthetic protocol included buprenorphine-propofol-isoflurane. Meloxicam [0.2 mg/kg body weight (BW)] was administered subcutaneously before surgery. Cats were randomly divided into 2 groups to receive 1 of 2 treatments. Intraperitoneal bupivacaine 0.25% (2 mg/kg BW) was administered with epinephrine (BE group; 2 μg/kg BW) or dexmedetomidine (BD group; 1 μg/kg BW) before ovariohysterectomy (n = 8/group). A catheter was placed in the jugular vein for blood sampling. Blood samples were collected for up to 8 h after bupivacaine was administered. Plasma concentrations and pharmacokinetics of bupivacaine were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS) and non-compartmental model, respectively. Pain was evaluated using the UNESP-Botucatu multidimensional composite pain scale (MCPS), the Glasgow composite feline pain scale (GPS), and a dynamic visual analog scale up to 8 h after extubation. Rescue analgesia was provided with buprenorphine if MCPS was ≥ 6. Repeated measures linear models were used for analysis of pain and sedation scores (P < 0.05). Maximum bupivacaine plasma concentrations (Cmax) for BE and BD were 1155 ± 168 ng/mL and 1678 ± 364 ng/mL (P = 0.29) at 67 ± 13 min (Tmax) and 123 ± 59 min (P = 0.17), respectively. Pharmacokinetic parameters and pain scores were not different between treatments (P > 0.05). One cat in the BE group received rescue analgesia (P = 0.30). Intraperitoneal bupivacaine with epinephrine or dexmedetomidine produced concentrations below toxic levels and similar analgesic effects. It is therefore safe to administer these drug combinations in cats undergoing ovariohysterectomy.

OBJECTIVE To identify whether age, sex, or breed is associated with crown height of the left and right maxillary first molar tooth (M1) measured on CT images, to develop a mathematical model to determine age of horses by use of M1 crown height, and to determine the correlation between M1 crown height measured on radiographic and CT images. SAMPLE CT (n = 735) and radiographic images (35) of the heads of horses. PROCEDURES Crown height of left and right M1 was digitally measured on axial CT views. Height was measured on a lateral radiographic image when available. Linear regression analysis was used to identify factors associated with crown height. Half the data set was subsequently used to generate a regression model to predict age on the basis of M1 crown height, and the other half was used to validate accuracy of the predictions. RESULTS M1 crown height decreased with increasing age, but the rate of decrease slowed with increasing age. Height also differed by sex and breed. The model most accurately reflected age of horses < 10 years old, although age was overestimated by a mean of 0.1 years. The correlation between radiographic and CT crown height of M1 was 0.91; the mean for radiographic measurements was 2.5 mm greater than for CT measurements. CONCLUSIONS AND CLINICAL RELEVANCE M1 crown height can be used to predict age of horses. Results for CT images correlated well with those for radiographic images. Studies are needed to develop a comparable model with results for radiographic images.

To obtain immunogenic conjugate antigens, adipic acid dihydrazide (ADH), as a bridge, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDAC), as a coupling agent, were used to conjugate the purified fusion protein, clumping factor A-fibronectin binding protein ClfA A-FnBPA, and type 5 capsular polysaccharide (CP5). The conjugates were mixed with an adjuvant, and mice were immunized 3 times and challenged with Staphylococcus aureus 1 week later. Antibody titers were determined by indirect enzyme-linked immunosorbent assay (ELISA). At 14 days after the first immunization, antibodies against the purified protein and conjugate were detected; after 28 days, antibody levels increased; and a week after the third immunization, antibody levels continued to increase. However, the conjugate antibody titers were higher than those of the purified protein during the study, and no IgG antibodies against purified CP5 were detected during the entire experiment. The protection rate increased to 90% in the conjugate group, indicating that the conjugate imparts a relatively higher protective efficacy than the purified protein and purified CP5.

OBJECTIVE To determine whether target values for pharmacokinetic-pharmacodynamic (PK-PD) indices against selected canine pathogens were achievable for pradofloxacin in various canine fluids and leukocytes. ANIMALS 8 healthy adult hounds (experiments 1 and 2) and 6 healthy adult dogs (experiment 3). PROCEDURES In 3 experiments, pradofloxacin (3, 6, or 12 mg/kg) and enrofloxacin (5 or 10 mg/kg) were orally administered once a day for 5 days, and blood, interstitial fluid (ISF), and other fluid samples were collected at various points. Sample drug concentrations were measured, and noncompartmental pharmacokinetic analysis was performed; then, PK-PD indices (ratios between maximum observed concentration [Cmax] and minimum inhibitory or mutant prevention concentrations) were determined for 7 bacterial species. RESULTS PK-PD values for pradofloxacin at 3 mg/kg were approximately 5 times as high in leukocyte versus plasma and were lowest in CSF, synovial fluid, and aqueous humor. No significant differences were noted between serum and ISF. Value ratios for serum versus other body fluids were numerically higher for pradofloxacin (vs enrofloxacin) for all fluid types except CSF and aqueous humor. Target PK-PD values were exceeded for pradofloxacin against all 7 bacterial species in leukocytes and against all species except Bacteroides spp in serum and ISF. Enrofloxacin achieved the target Cmax-to-minimum inhibitory concentration ratio against Pasteurella multocida in serum, ISF, and leukocytes and for Staphylococcus pseudintermedius in serum and leukocytes. A Cmax-to-mutant prevention concentration ratio ≥ 1 against Eschericha coli was achieved for pradofloxacin at 6 mg/kg. CONCLUSIONS AND CLINICAL RELEVANCE These findings supported once-daily oral administration of pradofloxacin to dogs at the currently recommended dose (7.5 mg/kg).

OBJECTIVE To evaluate effects of 2 levels of partial neuromuscular block on the ventilatory response to a hypercapnic challenge in anesthetized dogs and to evaluate effects of edrophonium for reversing partial neuromuscular block. ANIMALS 6 healthy adult Beagles. PROCEDURES Each dog was anesthetized twice with propofol and dexmedetomidine. End-tidal partial pressure of CO2 (P(etco2)), tidal volume (V(t)), and peak inspiratory flow (PIF) were measured during breathing at rest. Maximal V(t) and PIF (V(tMAX) and PIF(MAX), respectively) in response to a hypercapnic challenge consisting of 10% CO2 inhaled for 1 minute were measured. Variables were measured before administration of atracurium (baseline), during moderate (train-of-four [TOF] ratio, 0.3 to 0.5) and mild (TOF ratio, 0.6 to 0.8) atracurium-induced neuromuscular block, and after neuromuscular block recovery (TOF ratio, ≥ 0.9) following administration of edrophonium or saline (0.9% NaCl) solution. Dogs for which any variable returned to < 80% of the baseline value were identified. RESULTS Partial neuromuscular block increased P(etco2); it impaired V(t) at rest and Vt(MAX) but not PIF at rest and PIF(MAX). All variables except P(etco2) returned to baseline values when the TOF returned to ≥ 0.9. After recovery from neuromuscular block, significantly more dogs had a V(tMAX) < 80% of the baseline value when edrophonium was not administered. CONCLUSIONS AND CLINICAL RELEVANCE Partial neuromuscular block in anesthetized Beagles decreased spontaneous ventilation at rest and impaired the response to a hypercapnic challenge. Response to hypercapnic challenge might remain partially impaired after recovery of the TOF ratio to ≥ 0.9.

The purpose of this study was to determine the pharmacokinetics of cannabidiol (CBD) in healthy dogs. Thirty, healthy research dogs were assigned to receive 1 of 3 formulations (oral microencapsulated oil beads, oral CBD-infused oil, or CBD-infused transdermal cream), at a dose of 75 mg or 150 mg q12h for 6 wk. Serial cannabidiol plasma concentrations were measured over the first 12 h and repeated at 2, 4, and 6 wk. Higher systemic exposures were observed with the oral CBD-infused oil formulation and the half-life after a 75-mg and 150-mg dose was 199.7 ± 55.9 and 127.5 ± 32.2 min, respectively. Exposure is dose-proportional and the oral CBD-infused oil provides the most favorable pharmacokinetic profile.

OBJECTIVE To determine effects of 3 plasma concentrations of fentanyl on the minimum alveolar concentration of isoflurane (MACiso) and cardiovascular variables in Hispaniolan Amazon parrots (Amazona ventralis). ANIMALS 6 adult parrots. PROCEDURES In phase 1, anesthesia was induced and maintained with isoflurane; intermittent positive-pressure ventilation was provided. The MACiso was determined for each bird by use of a bracketing method and supramaximal electrical stimulus. Fentanyl (20 μg/kg) was administered IV, and blood samples were collected over time to measure plasma fentanyl concentrations for pharmacokinetic calculations. In phase 2, pharmacokinetic values for individual birds were used for administration of fentanyl to achieve target plasma concentrations of 8, 16, and 32 ng/mL. At each concentration, MACiso and cardiovascular variables were determined. Data were analyzed with mixed-effects multilevel linear regression analysis. RESULTS Mean ± SD fentanyl plasma concentrations were 0 ng/mL, 5.01 ± 1.53 ng/mL, 12.12 ± 3.58 ng/mL, and 24.93 ± 4.13 ng/mL, and MACiso values were 2.09 ± 0.17%, 1.45 ± 0.32%, 1.34 ± 0.31%, and 0.95 ± 0.14% for fentanyl target concentrations of 0, 8, 16, and 32 ng/mL, respectively. Fentanyl significantly decreased MACiso in a dose-dependent manner. Heart rate and blood pressure significantly decreased at all fentanyl doses, compared with values for MACiso at 0 ng of fentanyl/mL. CONCLUSIONS AND CLINICAL RELEVANCE Fentanyl significantly decreased the MACiso in healthy Hispaniolan Amazon parrots, but this was accompanied by a depressive effect on heart rate and blood pressure that would need to be considered for application of this technique in clinical settings.

OBJECTIVE To determine antinociceptive efficacy, behavioral patterns, and respiratory effects associated with dexmedetomidine administration in ball pythons (Python regius). ANIMALS 12 ball pythons. PROCEDURES Antinociception was assessed by applying an infrared heat stimulus to the cranioventral surface of snakes during 2 experiments. Thermal withdrawal latency was measured at 0, 2, and 24 hours after SC injections of dexmedetomidine (0.1 or 0.2 mg/kg) or saline (0.9% NaCl) solution and at 0 to 60 minutes after injection of dexmedetomidine (0.1 mg/kg) or saline solution. Behaviors were recorded at 0, 2, and 24 hours after administration of dexmedetomidine (0.1 mg/kg) or saline solution. Tongue flicking, head flinch to the approach of an observer's hand, movement, and righting reflex were scored. Respiratory frequency was measured by use of plethysmography to detect breathing-related movements after injection of dexmedetomidine (0.1 mg/kg) or saline solution. RESULTS Mean baseline withdrawal latency was 5 to 7 seconds; saline solution did not alter withdrawal latency. Dexmedetomidine increased withdrawal latency by 18 seconds (0.2 mg/kg) and 13 seconds (0.1 mg/kg) above baseline values at 2 hours. Increased withdrawal latency was detected within 15 minutes after dexmedetomidine administration. At 2 hours after injection, there were few differences in behavioral scores. Dexmedetomidine injection depressed respiratory frequency by 55% to 70%, compared with results for saline solution, but snakes continued to breathe without prolonged apnea. CONCLUSIONS AND CLINICAL RELEVANCE Dexmedetomidine increased noxious thermal withdrawal latency without causing excessive sedation. Therefore, dexmedetomidine may be a useful analgesic drug in ball pythons and other snake species.

OBJECTIVE To assess pharmacokinetics of tranexamic acid (TXA) in dogs and assess antifibrinolytic properties of TXA in canine blood by use of a thromboelastography-based in vitro model of hyperfibrinolysis. ANIMALS 6 healthy adult dogs. PROCEDURES Dogs received each of 4 TXA treatments (10 mg/kg, IV; 20 mg/kg, IV; approx 15 mg/kg, PO; and approx 20 mg/kg, PO) in a randomized crossover-design study. Blood samples were collected at baseline (time 0; immediately prior to drug administration) and predetermined time points afterward for pharmacokinetic analysis and pharmacodynamic (thromboelastography) analysis by use of an in vitro hyperfibrinolysis model. RESULTS Maximum amplitude (MA [representing maximum clot strength]) significantly increased from baseline at all time points for all treatments. The MA was lower at 360 minutes for the 10-mg/kg IV treatment than for other treatments. Percentage of clot lysis 30 minutes after MA was detected was significantly decreased from baseline at all time points for all treatments; at 360 minutes, this value was higher for the 10-mg/kg IV treatment than for other treatments and higher for the 20-mg/kg IV treatment than for the 20-mg/kg PO treatment. Maximum plasma TXA concentrations were dose dependent. At 20 mg/kg, IV, plasma TXA concentrations briefly exceeded concentrations suggested for complete inhibition of fibrinolysis. Oral drug administration resulted in a later peak antifibrinolytic effect than did IV administration. CONCLUSIONS AND CLINICAL RELEVANCE Administration of TXA improved clot strength and decreased fibrinolysis in blood samples from healthy dogs in an in vitro hyperfibrinolysis model. Further research is needed to determine clinical effects of TXA in dogs with hyperfibrinolysis.