Objective—To determine effects of various concentrations of retinoic acid (RA) or a synthetic RA receptor antagonist (LE135) on equine chondrocytes or bone marrow—derived equine mesenchymal stem cells (BMDMSCs) in monolayer cultures. Sample—Articular cartilage and BMDMSCs from 5 clinically normal horses. Procedures—Monolayers of chondrocytes cultured in standard media and of BMDMSCs cultured in chondrogenic media were treated with RA at concentrations of 0, 0.1, 1, or 10μM or LE135 at concentrations of 0, 0.1, 1, or 10μM on day 0. On days 7 and 14, samples were analyzed for DNA concentration, chondrocyte morphology or features consistent with chondrogenesis (ie, chondral morphology [scored from 0 to 4]), and gene expression of collagen type Ia (CI), collagen type II (CII), and aggrecan. Results—Chondrocytes treated with RA had more mature chondral morphology (range of median scores, 3.0 to 4.0) than did untreated controls (range of median scores, 0.5 to 0.5). Chondrocytes treated with LE135 did not sustain chondrocyte morphology. All BMDMSCs had evidence of chondral morphology or high CII:CI ratio. Retinoic acid (1 or 10μM) or LE135 (10μM) treatment decreased DNA content of BMDMSC cultures. At 0.1 and 1μM concentrations, LE135 weakly but significantly increased chondral morphology scores, compared with untreated controls, but lack of aggrecan expression and lack of increased CII:CI ratio, compared with that of controls, did not affect chondrogenesis. Conclusions and Clinical Relevance—RA promoted maturation and hypertrophy in chondrocytes but not BMDMSCs in monolayer cultures. Deficiency or blockade of RA may prevent hypertrophy and maturation of differentiated chondrocytes.

This study assessed the feasibility of measuring tiludronate in horses using a minimally invasive bone biopsy technique. Eight horses were treated with intravenous (IV) tiludronate [1 mg/kg bodyweight (BW)], either once (n = 4) or twice, 28 d apart (n = 4). The horses that were treated once were euthanized on days 1, 43, 57, or 92 and those that were treated twice, were euthanized on days 112, 154, 194, or 364. Bone samples were taken bilaterally from each horse at 4 sites: the third metacarpal bone (MCIII), the 13th rib (R13), the tuber coxae (TC), and the cuboid bone (CB). Test samples were taken with a 5-mm diameter dental drill, while larger reference samples were taken with an osteotome. The concentrations of tiludronate were measured by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The TC was the easiest site to sample, and no technical difficulties were encountered for extraction and measurement. Drill sampling at the MCIII was difficult. Moreover, both the extraction and measurement caused technical problems and results were unreliable in most cases (93%). Drill samples obtained from the R13 were very small and access to the CB required considerable dissection, which would not be feasible in vivo. Forty-six percent and 36% of the tiludronate measurements performed on the R13 and CB samples, respectively, were unreliable. The ratio of tiludronate concentrations ranged from 73% to 185% (median: 118%) in the TC, 65% to 208% (median: 81%) in the R13, and 26% to 110% (median: 57%) in the CB. In all but 1 horse, the highest concentrations of tiludronate were found in the TC. It was concluded that bone biopsies performed at the TC were adequate for measuring tiludronate in horses and should be considered in future for repeated measurements over time in living animals.

Objective: To compare computed tomography (CT) images of equine tarsi with cross-sectional anatomic slices and evaluate the potential of CT for imaging pathological tarsal changes in horses. Sample: 6 anatomically normal equine cadaveric hind limbs and 4 tarsi with pathological changes. Procedures: Precontrast CT was performed on 3 equine tarsi; sagittal and dorsal reconstructions were made. In all limbs, postcontrast CT was performed after intra-articular contrast medium injection of the tarsocrural, centrodistal, and tarsometatarsal joints. Images were matched with corresponding anatomic slices. Four tarsi with pathological changes underwent CT examination. Results: The tibia, talus, calcaneus, and central, fused first and second, third, and fourth tarsal bones were clearly visualized as well as the long digital extensor, superficial digital flexor, lateral digital flexor (with tarsal flexor retinaculum), gastrocnemius, peroneus tertius, and tibialis cranialis tendons and the long plantar ligament. The lateral digital extensor, medial digital flexor, split peroneus tertius, and tibialis cranialis tendons and collateral ligaments could be located but not always clearly identified. Some small tarsal ligaments were identifiable, including plantar, medial, interosseus, and lateral talocalcaneal ligaments; interosseus talocentral, centrodistal, and tarsometatarsal ligaments; proximal and distal plantar ligaments; and talometatarsal ligament. Parts of the articular cartilage could be assessed on postcontrast images. Lesions were detected in the 4 tarsi with pathological changes. Conclusions and Clinical Relevance: CT of the tarsus is recommended when radiography and ultrasonography are inconclusive and during preoperative planning for treatment of complex fractures. Images from this study can serve as a CT reference, and CT of pathological changes was useful.

Objective—To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies. Sample—Human, canine, and equine liver microsomes and human single CYP3A4 and CYP2C9 and their canine orthologs. Procedures—Chemical inhibitors selective for human CYP enzymes and anti-CYP antibodies were incubated with racemic ketamine and liver microsomes or specific CYPs. Ketamine N-demethylation to norketamine was determined via enantioselective capillary electrophoresis. Results—The general CYP inhibitor 1-aminobenzotriazole almost completely blocked ketamine metabolism in human and canine liver microsomes but not in equine microsomes. Chemical inhibition of norketamine formation was dependent on inhibitor concentration in most circumstances. For all 3 species, inhibitors of CYP3A4, CYP2A6, CYP2C19, CYP2B6, and CYP2C9 diminished N-demethylation of ketamine. Anti-CYP3A4, anti-CYP2C9, and anti-CYP2B6 antibodies also inhibited ketamine N-demethylation. Chemical inhibition was strongest with inhibitors of CYP2A6 and CYP2C19 in canine and equine microsomes and with the CYP3A4 inhibitor in human microsomes. No significant contribution of CYP2D6 to ketamine biotransformation was observed. Although the human CYP2C9 inhibitor blocked ketamine N-demethylation completely in the canine ortholog CYP2C21, a strong inhibition was also obtained by the chemical inhibitors of CYP2C19 and CYP2B6. Ketamine N-demethylation was stereoselective in single human CYP3A4 and canine CYP2C21 enzymes. Conclusions and Clinical Relevance—Human-specific inhibitors of CYP2A6, CYP2C19, CYP3A4, CYP2B6, and CYP2C9 diminished ketamine N-demethylation in dogs and horses. To address drug-drug interactions in these animal species, investigations with single CYPs are needed.

Objective: To determine the impact of a free-choice diet on nutritional intake and body condition of feral horses. Animals: Cadavers of 41 feral horses from 5 Australian locations. Procedures: Body condition score (BCS) was determined (scale of 1 to 9), and the stomach was removed from horses during postmortem examination. Stomach contents were analyzed for nutritional variables and macroelement and microelement concentrations. Data were compared among the locations and also compared with recommended daily intakes for horses. Results: Mean BCS varied by location; all horses were judged to be moderately thin. The BCS for males was 1 to 3 points higher than that of females. Amount of protein in the stomach contents varied from 4.3% to 14.9% and was significantly associated with BCS. Amounts of water-soluble carbohydrate and ethanol-soluble carbohydrate in stomach contents of feral horses from all 5 locations were higher than those expected for horses eating high-quality forage. Some macroelement and microelement concentrations were grossly excessive, whereas others were grossly deficient. There was no evidence of ill health among the horses. Conclusions and Clinical Relevance: Results suggested that the diet for several populations of feral horses in Australia appeared less than optimal. However, neither low BCS nor trace mineral deficiency appeared to affect survival of the horses. Additional studies on food sources in these regions, including analysis of water-soluble carbohydrate, ethanol-soluble carbohydrate, and mineral concentrations, are warranted to determine the provenance of such rich sources of nutrients. Determination of the optimal diet for horses may need revision.

Objective—To induce ischemia and reperfusion injury in the large colon mucosa of horses in vivo and evaluate the recovery and effects of components of an organ transplant solution on mucosal recovery in vitro. Animals—6 healthy horses. Procedures—Horses were anesthetized, and ischemia was induced for 60 minutes in the pelvic flexure, which was followed by reperfusion for 240 minutes. Ischemic (n = 4 horses), reperfused (6), and adjacent control (6) colonic mucosae were isolated for in vitro testing and histologic examinations. Tissues were mounted in Ussing chambers with plain Krebs Ringer bicarbonate (KRB), KRB with N-acetylcysteine (NAC), or KRB with a modified organ transplant solution (MOTS). Transepithelial electrical resistance (TER) and mannitol flux were used to assess mucosal integrity. Data were analyzed by use of ANOVA and Kruskal-Wallis tests. Results—The TER in reperfused tissues was similar to the TER in control tissues and greater than the TER in ischemic tissues, which was consistent with morphological evidence of recovery in reperfused tissues. Mannitol flux was greater in ischemic tissues than in reperfused tissues. The TER and mannitol flux were not significantly affected by incubation of mucosa with NAC or MOTS. Conclusions and Clinical Relevance—Ischemia induced during the brief period allowed rapid mucosal repair and complete recovery of tissue barrier properties during reperfusion. Therefore, reperfusion injury was not observed for this method of ischemic damage in equine colonic mucosa.

Objective—To evaluate a commercially available modified-live Streptococcus equi subsp equi vaccine for safety and persistence in vaccinated ponies and to detect recombination or reversion events in the vaccine strain. Animals—5 ponies that were 1.5 to 8 years old (group 1) and 4 ponies that were 6 months old (group 2). Procedures—Ponies were vaccinated, with a subsequent booster vaccination 2 to 3 weeks later, and monitored for 50 days. At booster vaccination, an equal amount of a tracycline-resistant wild-type strain of S equi was administered. Recovery of all strains was performed by use of bacteriologic culture and PCR assays. Results—Ponies in group 1 had background antibody titers against S equi antigen before vaccination despite the lack of known exposure to S equi. Ponies in group 2 were immunologically naïve. Increases in anti-S equi antibody titers were detected in both groups. Ponies in group 1 did not have clinical signs of disease caused by S equi. In group 2, all ponies developed abscesses in retropharyngeal lymph nodes; 1 pony developed severe clinical disease and was euthanized. The vaccine strain was recovered from ponies in group 2 for up to 24 days after vaccination. Conclusions and Clinical Significance—Although the vaccine was successful in inducing IgG antibodies against S equi in all ponies, findings suggested that the vaccine may have caused substantial morbidity and some deaths in the young ponies. In young ponies, the vaccine strain persisted in tissues for weeks; however, no evidence of recombination was detected.

Objective--To test the hypotheses that preparation method, exposure to shear force, and exposure to collagen affect the release of growth factors from equine platelet-rich plasma (PRP). Sample Population--PRP obtained from 6 horses. Procedures--PRP was prepared via 2 preparation methods (tube and automated) and subjected to 6 treatment conditions (resting, detergent, exposure to shear via 21- and 25-gauge needles, and exposure to collagen [10 and 20 μg/mL]). Concentrations of platelet-derived growth factor, isoform BB (PDGF-BB); transforming growth factor β, isoform 1 (TGFβ1); and insulin-like growth factor, isoform 1 (IGF-1) were quantified by use of ELISAs. Statistical analysis was conducted via repeated-measures ANOVA. Results--Platelet numbers were significantly higher in tube-prepared PRP than in automated-prepared PRP Growth factor concentrations did not differ significantly between preparation methods. Mean PDGF-BB concentration ranged from 134 to 7,157 pg/mL, mean TGFβ1 concentration ranged from 1,153 to 22,677 pg/mL, and mean IGF-1 concentration ranged from 150 to 280 ng/mL. Shear force did not affect growth factor concentrations. Dose-dependent increases in PDGF-BB and TGFβ1 were detected in response to collagen, but equalled only 10% of the estimated total platelet content. Concentrations of IGF-1 were not significantly different among treatments and negative or positive control treatments. Serum concentrations of PDGF-BB and TGFβ1 exceeded concentrations in PRP for most treatment conditions. Conclusions and Clinical Relevance--Release of growth factors from equine PRP was negligible as a result of the injection process alone. Investigation of platelet-activation protocols is warranted to potentially enhance PRP treatment efficacy in horses.

Objective—To determine the effect of differences in structural and mechanical tendon properties on functionality of the passive stay apparatus in horses. Sample—5 forelimbs each from nondwarf Friesians, dwarf Friesians, and ponies. Procedures—Harvested forelimbs were loaded to test the passive stay apparatus. Tendons that stabilize the distal portion of the limb (superficial digital flexor tendon, deep digital flexor tendon, and tendo interosseus [suspensory ligament]) were isolated, and force-elongation data were obtained. Bone lengths, initial tendon lengths, and initial tendon cross-sectional areas were measured, and Young moduli were calculated. A model was used to determine whether joint angles could be explained by these 4 factors only. Results—Dwarf limbs were unable to stand passively under loading because tendons that prevent overextension of the distal limb joints were too long and compliant to prevent over-extension. Tendon properties of limbs of nondwarf Friesians appeared to be intermediate between those of ponies and dwarf Friesians. Conclusions and Clinical Relevance—Dysfunction of the passive stay apparatus in dwarf Friesians could be related to differences in structural and material properties of the tendons that result in hyperextension of the joints under loading. Nondwarf Friesians had intermediate tendon properties, which might be a breed-specific variation. Results indicated that certain tendon properties were associated with load failure of the stay apparatus and provided additional information about the functionality and requirements of the passive stay apparatus.

Objective—To determine effects of a continuous rate infusion of lidocaine on the minimum alveolar concentration (MAC) of sevoflurane in horses. Animals—8 healthy adult horses. Procedures—Horses were anesthetized via IV administration of xylazine, ketamine, and diazepam; anesthesia was maintained with sevoflurane in oxygen. Approximately 1 hour after induction, sevoflurane MAC determination was initiated via standard techniques. Following sevoflurane MAC determination, lidocaine was administered as a bolus (1.3 mg/kg, IV, over 15 minutes), followed by constant rate infusion at 50 μg/kg/min. Determination of MAC for the lidocaine-sevoflurane combination was started 30 minutes after lidocaine infusion was initiated. Arterial blood samples were collected after the lidocaine bolus, at 30-minute intervals, and at the end of the infusion for measurement of plasma lidocaine concentrations. Results—IV administration of lidocaine decreased mean ± SD sevoflurane MAC from 2.42 ± 0.24% to 1.78 ± 0.38% (mean MAC reduction, 26.7 ± 12%). Plasma lidocaine concentrations were 2,589 ± 811 ng/mL at the end of the bolus; 2,065 ± 441 ng/mL, 2,243 ± 699 ng/mL, 2,168 ± 339 ng/mL, and 2,254 ± 215 ng/mL at 30, 60, 90, and 120 minutes of infusion, respectively; and 2,206 ± 329 ng/mL at the end of the infusion. Plasma concentrations did not differ significantly among time points. Conclusions and Clinical Relevance—Lidocaine could be useful for providing a more balanced anesthetic technique in horses. A detailed cardiovascular study on the effects of IV infusion of lidocaine during anesthesia with sevoflurane is required before this combination can be recommended.