OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro. SAMPLE Venous blood samples from 40 adult horses. PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities. RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

OBJECTIVE To characterize the MRI and histologic features of the supraspinatus tendon in nonlame dogs. ANIMALS 7 cadavers (14 shoulder joints) of nonlame 2-year-old sexually intact male Beagles. PROCEDURES Multiple MRI fluid-sensitive pulse sequences were obtained for both shoulder joints of each cadaver, and the thickness, volume, and signal intensity of each supraspinatus tendon were assessed. After MRI scanning was complete, the shoulder joints were processed for histologic examination. Tissue specimens were stained with various stains to determine tendon morphology and composition. Histologic and MRI findings were correlated and described. RESULTS All supraspinatus tendons had a trilaminar appearance on sagittal and transverse MRI images, which was characterized by a thick, hyperintense center layer (central substance) sandwiched between thin hypointense superficial and deep margins. The mean ± SD central substance-to-superficial margin and central substance-to-deep margin thickness ratios were 8.4 ± 1.2 and 9.0 ± 0.9, respectively; supraspinatus tendon-to-triceps brachii muscle signal intensity ratio was 1.3 ± 0.2; and tendon volume was 445 ± 20 mm3. The superficial and deep margins histologically resembled other tendons with highly ordered collagen fibers. The central substance was comprised of water-rich glycosaminoglycans interspersed among haphazardly arranged collagen bundles. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated histologically normal canine supraspinatus tendons have a trilaminar appearance on MRI images. In dogs, a diagnosis of supraspinatus tendinosis should not be based solely on the tendon having a hyperintense signal on MRI images; other MRI evidence of shoulder joint disease and diagnostic findings are necessary to support such a diagnosis.

OBJECTIVE To determine the physiochemical properties and pharmacokinetics of 3 midazolam gel formulations following buccal administration to dogs. ANIMALS 5 healthy adult hounds. PROCEDURES In phase 1 of a 2-phase study, 2 gel formulations were developed that contained 1% midazolam in a poloxamer 407 (P1) or hydroxypropyl methylcellulose (H1) base and underwent rheological and in vitro release analyses. Each formulation was buccally administered to 5 dogs such that 0.3 mg of midazolam/kg was delivered. Each dog also received midazolam hydrochloride (0.3 mg/kg, IV). There was a 3-day interval between treatments. Blood samples were collected immediately before and at predetermined times for 8 hours after drug administration for determination of plasma midazolam concentration and pharmacokinetic analysis. During phase 2, a gel containing 2% midazolam in a hydroxypropyl methylcellulose base (H2) was developed on the basis of phase 1 results. That gel was buccally administered such that midazolam doses of 0.3 and 0.6 mg/kg were delivered. Each dog also received midazolam (0.3 mg/kg, IV). All posttreatment procedures were the same as those for phase 1. RESULTS The H1 and H2 formulations had lower viscosity, greater bioavailability, and peak plasma midazolam concentrations that were approximately 2-fold as high, compared with those for the P1 formulation. The mean peak plasma midazolam concentration for the H2 formulation was 187.0 and 106.3 ng/mL when the midazolam dose administered was 0.6 and 0.3 mg/kg, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that buccal administration of gel formulations might be a viable alternative for midazolam administration to dogs.

OBJECTIVE To evaluate effects of high-concentration buprenorphine (HCB) on self-injurious behavior, food intake, fecal output, and thermal withdrawal latencies in healthy rats. ANIMALS 8 Sprague-Dawley rats. PROCEDURES Rats received 4 SC treatments (HCB at 0.075, 0.15, or 0.30 mg/kg [HCB0.075, HCB0.15, and HCB0.30, respectively] or 5% dextrose solution [0.20 mL/kg]) in a randomized, crossover-design study. Self-injurious behavior was assessed for 8 hours after injection. Food intake and fecal output were assessed for predetermined periods before and after treatment and separated into 12-hour light and dark periods for further analysis. Withdrawal latencies were assessed before (time 0) and at predetermined times after injection. Data were compared among treatments and time points. RESULTS Self-injurious behavior was observed up to 8 hours after injection for all HCB, but not dextrose, treatments. Preinjection food intake and fecal output amounts were similar among groups and higher during the dark period than during the light period. Food intake after all HCB treatments was higher during the light period and lower during the dark period, compared with preinjection results for the same treatments and with postinjection results for dextrose administration. Light-period fecal output was lower after HCB0.15 and HCB0.30 administration, compared with preinjection values for the same treatments and postinjection values for dextrose administration. Percentage change in withdrawal latency was significantly higher than that at time 0 (ie, 0%) for only 1 treatment (HCB0.30) at 1 time point (1 hour after injection). CONCLUSIONS AND CLINICAL RELEVANCE Although HCB0.30 produced a degree of thermal hypoalgesia in healthy rats, self-injurious behavior and alterations in food intake and fecal output were detected, potentially affecting clinical utility of the treatment.

OBJECTIVE To determine from MRI measurements whether soft palate length (SPL) and thickness are correlated in dogs, evaluate the association between the olfactory bulb angle (OBA) and degree of brachycephalia, and determine the correlation between soft palate–epiglottis overlap and OBA in dogs. ANIMALS 50 brachycephalic and 50 nonbrachycephalic client-owned dogs without abnormalities of the head. PROCEDURES Medical records and archived midsagittal T2-weighted MRI images of brachycephalic and nonbrachycephalic dogs' heads were reviewed. Group assignment was based on breed. Data collected included weight, SPL and thickness, OBA, and the distance between the caudal extremity of the soft palate and the basihyoid. Soft palate length and thickness were adjusted on the basis of body weight. RESULTS Brachycephalic dogs had significantly thicker soft palates and lower OBAs, compared with findings for nonbrachycephalic dogs. There was a significant negative correlation (r2 = 0.45) between OBA and soft palate thickness. The correlation between SPL and OBA was less profound (r2 = 0.09). The distance between the caudal extremity of the soft palate and the basihyoid was shorter in brachycephalic dogs than in nonbrachycephalic dogs. The percentage of epiglottis–soft palate overlap significantly decreased with increasing OBA (r2 = 0.31). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MRI images can be consistently used to assess anatomic landmarks for measurement of SPL and thickness, OBA, and soft palate-to-epiglottis distance in brachycephalic and nonbrachycephalic dogs. The percentage of epiglottis–soft palate overlap was significantly greater in brachycephalic dogs and was correlated to the degree of brachycephalia.

Foot-and-mouth disease (FMD) and vesicular stomatitis (VS) cause such similar clinical signs and lesions that laboratory tests are required to distinguish between infections caused by each virus. Using mouse anti-foot-and-mouth disease virus (FMDV) 3B monoclonal or polyclonal anti-vesicular stomatitis virus-New Jersey (VSV-NJ) antibodies and recombinant FMDV 3ABC or VSV-NJ glycoprotein (G) antigens coated to MagPlex beads, competitive Luminex immunoassays (cLIAs) were developed for FMDV and VSV-NJ, respectively. The cLIAs successfully detected antibodies to FMDV 3ABC and VSV-NJ G in sera from infected animals. The diagnostic sensitivity and specificity were 93% and 98%, respectively for FMDV and 93% and 95.4%, respectively for VSV-NJ. These cLIAs are potential alternatives for competitive enzyme-linked immunosorbent assays (cELISAs) and provide the opportunity for multiplexing to reduce time and the amount of serum required for testing.

The objective of this study was to examine the effects of immunosuppressive prednisolone therapy on pancreatic tissue and the concentration of serum canine pancreatic lipase immunoreactivity (cPLI) in healthy dogs. Six healthy beagle dogs were subcutaneously administered an immunosuppressive dose of prednisolone [4 mg/kg body weight (BW)] once daily for either 2 or 3 weeks. Serum cPLI concentration was measured before and after treatment. Ultrasonographic examination of the pancreas and laparoscopic biopsy and histopathological examination of the right pancreatic lobe and the liver were also conducted before and after treatment. The expression of pancreatic lipase messenger ribonucleic acid (mRNA) in the pancreas and liver was examined by polymerase chain reaction (PCR). Although the serum cPLI concentration was significantly higher on day 14 and on the day of the second laparoscopy than before treatment, it was classified as normal (≤ 200 μg/L) in 5 dogs and as abnormal (≥ 400 μg/L) in only 1 dog. None of the 6 dogs showed clinical signs of pancreatitis during the study period. After treatment, ultrasonographic examination of the pancreas showed no changes except for a hypoechoic pancreas in 1 dog. Histopathological examination of the right pancreatic lobe in all dogs showed no evidence of pancreatitis after treatment. Pancreatic lipase mRNA expression was detected in the pancreas, but not in the liver, before and after treatment. The administration of 4 mg/kg BW per day of prednisolone for 2 or 3 weeks increased the serum cPLI concentration without clinical signs of pancreatitis, although an abnormal cPLI concentration (≥ 400 μg/L) was observed in only 1 dog. No ultrasonographic or histological evidence of pancreatitis was observed in any of the dogs.

The purpose of this study was to evaluate the effects of a single intravenous dose of alfaxalone on canine splenic volume. In 6 adult beagle dogs the splenic volume [mean ± standard error (SE)] was determined by computed tomography to be 0.17 ± 0.02 L before alfaxalone administration and 0.24 ± 0.02 L (P = 0.0091) and 0.23 ± 0.02 L (P = 0.0268) 15 and 30 min, respectively, after alfaxalone administration. Hematocrits (mean ± SE) obtained at the same times were, respectively, 46.3% ± 1.3%, 40.6% ± 1.3% (P = 0.0015), and 41.7% ± 1.3% (P = 0.0057). In conclusion, alfaxalone caused relaxation of the canine splenic capsule and an increase in the splenic volume, along with a decrease in the hematocrit in these dogs.

OBJECTIVE To evaluate the effects of drinking nutrient-enriched water (NW) on water intake and indices of hydration in healthy domestic cats fed a dry kibble diet ad libitum. ANIMALS 18 domestic shorthair cats. PROCEDURES Group-housed cats were assigned to tap water (TW; n = 9) or NW (9) groups. All cats received TW at baseline (days −7 to −1). No changes were made to the food-water regimen for the TW group. The NW group received NW instead of TW from days 0 through 10, then received TW and NW in separate bowls (days 11 through 56). Food intake was measured through day 10; liquid consumed by drinking was measured throughout the study. Blood and urine samples were collected at predetermined times for analyses; 48-hour urine collection (days 28 through 30 or 31 through 33) was performed to assess output volume and aid endogenous creatinine-based glomerular filtration rate (GFR) determination. Data were analyzed with linear mixed-effects models. RESULTS Baseline TW and calorie intake were similar between groups. The NW treatment was significantly associated with increased liquid consumption during the treatment phase. Mean urine output was significantly higher in the NW group (15.2 mL/kg/d) than in the TW group (10.3 mL/kg/d). Mean GFR (1.75 vs 1.87 mL/min/kg, respectively) did not differ between groups. Effects of treatment and time were each significant for urine specific gravity and osmolality and urine creatinine, phosphate, and urea nitrogen concentrations, with lower values for the NW group. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that consumption of the NW can increase liquid intake and improve measures of hydration in healthy cats. These effects may offer health benefits to some cats in need of greater water consumption.

OBJECTIVE To evaluate the microbial integrity of preservative-free cyclodextrin-based alfaxalone in a multiple-use system. SAMPLE 22 vials of preservative-free alfaxalone. PROCEDURES 2 storage conditions (room temperature, 22°C; refrigerated temperature, 4°C) and 3 handling techniques (closed system transfer device, nonclosed dispensing pin, and manufacturer-supplied vial stopper) comprised 6 treatment groups (3 replicates/group). An aliquot (0.5 mL) was withdrawn from each vial daily for 14 days. Samples were immediately inoculated into tryptic soy broth and incubated at 36°C for 24 hours; samples were subcultured onto 5% Columbia sheep blood agar and incubated for 48 hours. Isolated colonies were evaluated for identification. RESULTS There was no evidence of microbial contamination of vials stored for 7 days in refrigeration and handled with a protected port (closed system transfer device or nonclosed dispensing pin). CONCLUSIONS AND CLINICAL RELEVANCE The US FDA prohibits the use of alfaxalone beyond 6 hours after the vial stopper is broached (punctured), as mandated for a preservative-free injectable medication. Findings for the study reported here supported the use of alfaxalone for 7 days when refrigerated and handled with a single puncture of the stopper by use of a protected port (closed system transfer device or nonclosed dispensing pin). This would appear to be a practical alternative for an injectable anesthetic. It would minimize drug waste and the subsequent environmental impact for disposal of unused drug and allow standardization of storage and handling protocols for alfaxalone use in veterinary practices across the United States.