Objective—To characterize the in vitro effects of
oxytocin, acepromazine, xylazine, butorphanol,
detomidine, dantrolene, isoproterenol, and terbutaline
on skeletal and smooth muscle from the
Animals—14 adult horses without digestive tract disease.
Procedure—Circular and longitudinal strips from
the skeletal and smooth muscle of the esophagus
were suspended in tissue baths, connected to
force-displacement transducers interfaced with a
physiograph, and electrical field stimulation was
applied. Cumulative concentration-response curves
were generated for oxytocin, acepromazine,
xylazine, detomidine, butorphanol, isoproterenol,
terbutaline, and dantrolene. Mean maximum twitch
amplitude for 3 contractions/min was recorded and
compared with predrug-vehicle values for the
skeletal muscle segments, and area under the
curve (AUC) for 3 contractions/min was compared
with predrug-vehicle values for the smooth muscle
Results—No drugs caused a significant change in
skeletal muscle response. In smooth muscle, isoproterenol,
terbutaline, and oxytocin significantly
reduced AUC in a concentration-dependent manner.
Maximum reduction in AUC was 69% at 10–4M for
isoproterenol, 63% at 10–5M for terbutaline, and
64% at 10–4M for oxytocin.
Conclusions and Clinical Relevance—Isoproterenol,
terbutaline, and oxytocin cause relaxation of the
smooth muscle portion of the esophagus. The clinical
relaxant effects on the proximal portion of the esophagus
reported of drugs such as oxytocin, detomidine,
and acepromazine may be the result of centrally mediated
mechanisms. (Am J Vet Res 2002;63:1732–1737)
Objective—To compare effects of oxytocin, acepromazine
maleate, xylazine hydrochloride-butorphanol
tartrate, guaifenesin, and detomidine hydrochloride
on esophageal manometric pressure in horses.
Animals—8 healthy adult horses.
Procedure—A nasogastric tube, modified with 3
polyethylene tubes that exited at the postpharyngeal
area, thoracic inlet, and distal portion of the
esophagus, was fitted for each horse. Amplitude,
duration, and rate of propagation of pressure waveforms
induced by swallows were measured at 5, 10,
20, 30, and 40 minutes after administration of oxytocin,
detomidine, acepromazine, xylazine-butorphanol,
guaifenesin, or saline (0.9% NaCl) solution.
Number of spontaneous swallows, spontaneous
events (contractions that occurred in the absence of
a swallow stimulus), and high-pressure events (sustained
increases in baseline pressure of > 10 mm
Hg) were compared before and after drug administration.
Results—At 5 minutes after administration, detomidine
increased waveform amplitude and decreased
waveform duration at the thoracic inlet. At 10 minutes
after administration, detomidine increased waveform
duration at the thoracic inlet. Acepromazine administration
increased the number of spontaneous events
at the thoracic inlet and distal portion of the esophagus.
Acepromazine and detomidine administration
increased the number of high-pressure events at the
thoracic inlet. Guaifenesin administration increased
the number of spontaneous events at the thoracic
inlet. Xylazine-butorphanol, detomidine, acepromazine,
and guaifenesin administration decreased the
number of spontaneous swallows.
Conclusions and Clinical Relevance—Detomidine,
acepromazine, and a combination of xylazine butorphanol
had the greatest effect on esophageal motility
when evaluated manometrically. Reduction in spontaneous
swallowing and changes in normal, coordinated
peristaltic activity are the most clinically relevant
effects. (Am J Vet Res 2002;63:1738–1744)
OBJECTIVE To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses.
SAMPLE Jugular venous blood samples from 24 adult horses.
PROCEDURES Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates.
RESULTS Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry.
CONCLUSIONS AND CLINICAL RELEVANCE ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.
Objective—To determine the isometric responses of isolated intrapulmonary bronchioles from cats with and without adult heartworm infection.
Animals—13 purpose-bred adult cats.
Procedures—Cats were infected with 100 third-stage larvae or received a sham inoculation, and the left caudal lung lobe was collected 278 to 299 days after infection. Isometric responses of intrapulmonary bronchiolar rings were studied by use of a wire myograph. Three cycles of contractions induced by administration of 10μM acetylcholine were followed by administration of the contractile agonists acetylcholine, histamine, and 5-hydroxy-tryptamine. To evaluate relaxation, intrapulmonary bronchiolar rings were constricted by administration of 10μM 5-hydroxytryptamine, and concentration-response curves were generated from administration of sodium nitroprusside, isoproterenol, and substance P.
Results—Compared with tissues from control cats, contractile responses to acetylcholine and 5-hydroxytryptamine were reduced in tissues from heartworm-infected cats. Relaxation to isoproterenol was significantly reduced in tissues from heartworm-infected cats. Relaxation to substance P was increased in tissues from heartworm-infected cats, but relaxation to sodium nitroprusside was unchanged.
Conclusions and Clinical Relevance—Results suggested that despite increased bronchiolar wall thickness in heartworm-infected cats, a hyperreactive response of the bronchiolar smooth muscle is not the primary mechanism of respiratory tract clinical signs. Reduced response of the airway to isoproterenol may indicate refractoriness to bronchiolar relaxation in heartworm-infected cats.
OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses.
SAMPLE Jugular and cephalic venous blood samples from 17 adult horses.
PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate–labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry.
RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs.
CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses.
To investigate the effects of recombinant equine IL-1β on function of equine endothelial colony-forming cells (ECFCs) in vitro.
ECFCs derived from peripheral blood samples of 3 healthy adult geldings.
Function testing was performed to assess in vitro wound healing, tubule formation, cell adhesion, and uptake of 1,1′-dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate–labeled acetylated low-density lipoprotein (DiI-Ac-LDL) by cultured ECFCs. Cell proliferation was determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Effects on function test results of different concentrations and exposure times of recombinant equine IL-1β were assessed.
Challenge of cultured ECFCs with IL-1β for 48 hours inhibited tubule formation. Continuous challenge (54 hours) with IL-1β in the wound healing assay reduced gap closure. The IL-1β exposure did not significantly affect ECFC adhesion, DiI-Ac-LDL uptake, or ECFC proliferation.
CONCLUSIONS AND CLINICAL RELEVANCE
These results suggested a role for IL-1β in the inhibition of ECFC function in vitro. Functional changes in ECFCs following challenge with IL-1β did not appear to be due to changes in cell proliferative capacity. These findings have implications for designing microenvironments for and optimizing therapeutic effects of ECFCs used to treat ischemic diseases in horses.
Objective—To characterize adiponectin protein complexes in lean and obese horses.
Animals—26 lean horses and 18 obese horses.
Procedures—Body condition score (BCS) and serum insulin activity were measured for each horse. Denaturing and native western blot analyses were used to evaluate adiponectin complexes in serum. A human ELISA kit was validated and used to quantify high–molecular weight (HMW) complexes. Correlations between variables were made, and HMW values were compared between groups.
Results—Adiponectin was present as a multimer consisting of HMW (> 720-kDa), low-molecular weight (180-kDa), and trimeric (90-kDa) complexes in serum. All complexes were qualitatively reduced in obese horses versus lean horses, but the percentage of complexes < 250 kDa was higher in obese versus lean horses. High–molecular weight adiponectin concentration measured via ELISA was negatively correlated with serum insulin activity and BCS and was lower in obese horses (mean ± SD, 3.6 ± 3.9 μg/mL), compared with lean horses (8.0 ± 4.6 μg/mL).
Conclusions and Clinical Relevance—HMW adiponectin is measurable via ELISA, and concentration is negatively correlated with BCS and serum insulin activity in horses. A greater understanding of the role of adiponectin in equine metabolism will provide insight into the pathophysiology of metabolic disease conditions.
To describe misoprostol pharmacokinetics and anti-inflammatory efficacy when administered orally or per rectum in endotoxin-challenged horses.
6 healthy geldings.
A randomized 3-treatment crossover design was performed with a minimum washout period of 28 days between treatment arms. Prior to endotoxin challenge (lipopolysaccharide, 30 ng/kg IV over 30 minutes), horses received misoprostol (5 µg/kg once) per os (M-PO) or per rectum (M-PR) or water as control (CON). Clinical parameters were evaluated and blood samples obtained to measure plasma misoprostol free acid concentration, leukocyte counts, and tumor necrosis factor-α (TNFα) and interleukin 6 (IL-6) leukocyte gene expression and serum concentrations.
In the M-PO treatment arm, maximum plasma concentration and area under the concentration-versus-time curve (mean ± SD) were higher (5,209 ± 3,487 pg/mL and 17,998,254 ± 13,194,420 h·pg/mL, respectively) and median (interquartile range) time to maximum concentration (25 min [18 to 34 min]) was longer than in the M-PR treatment arm (854 ± 855 pg/mL; 644,960 ± 558,866 h·pg/mL; 3 min [3 to 3.5 min]). Significant differences in clinical parameters, leukocyte counts, and TNFα or IL-6 gene expression or serum protein concentration were not detected. Downregulation of relative gene expression was appreciated for individual horses in the M-PO and M-PR treatment arms at select time points.
Considerable variability in measured parameters was detected among horses within and between treatment arms. Misoprostol absorption and systemic exposure after PO administration differed from previous reports in horses not administered LPS. Investigation of multidose administration of misoprostol is warranted to better evaluate efficacy as an anti-inflammatory therapeutic.
To compare the pharmacokinetics between repeated doses and to characterize changes in the fecal microbiome after oral and rectal multidose misoprostol administration.
6 healthy university-owned geldings.
In a randomized, crossover study, misoprostol (5 μg/kg) was administered orally or rectally every 8 hours for 10 doses, or not administered (control), with a 21-day washout between treatments. Concentration-versus-time data for dose 1 and dose 10 were subject to noncompartmental analysis. For microbiota analysis using 16S rRNA amplicon sequencing, manure was collected 7 days before study onset, immediately before dose 1, and 6 hours, 7 days, and 14 days after dose 10, with time-matched points in controls.
Repeated dosing-related differences in pharmacokinetic parameters were not detected for either administration route. The area under the concentration-versus-time curve was greater (P < .04) after oral versus rectal administration. The relative bioavailability of rectal administration was 4 to 86% of that of oral administration. Microbial composition, richness, and β-diversity differed among subjects (P < .001 all) while only composition differed between treatments (P ≤ .01). Richness was decreased 6 hours after dose 10 and at the control-matched time point (P = .0109) in all subjects. No other differences for time points, treatments, or their interactions were observed.
Differences in systemic exposure were associated with the route of administration but were not detected after repeated administration of misoprostol. Differences in microbiota parameters were primarily associated with interindividual variation and management rather than misoprostol administration.