In vivo and in vitro effects of neostigmine on gastrointestinal tract motility of horses

Jorge E. Nieto Comparative Gastrointestinal Laboratory, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Betina Morales Comparative Gastrointestinal Laboratory, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Sawsan Z. Yamout Comparative Gastrointestinal Laboratory, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Scott D. Stanley California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Faye A. Harmon Comparative Gastrointestinal Laboratory, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Jack R. Snyder Comparative Gastrointestinal Laboratory, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Abstract

Objective—To determine the response to neostigmine of the contractile activity of the jejunum and pelvic flexure and the effects of a continuous rate infusion (CRI) of neostigmine in horses.

Animals—7 adult horses and tissue from 12 adult horses.

Procedures—A CRI of neostigmine (0.008 mg/kg/h) or placebo was administered to 6 horses in a crossover study design. Gastric emptying was evaluated by the acetaminophen test. The frequency of defecation and urination and the consistency and weight of feces were recorded throughout the experiment. The effect of neostigmine on smooth muscle contractile activity was evaluated in tissues from the jejunum and pelvic flexure. The effect of neostigmine and acetylcholine after incubation with muscarinic receptor antagonists (atropine and DAU 5884) and an acetylcholinesterase inhibitor (edrophonium) was also investigated in vitro.

Results—No difference was observed between neostigmine and placebo for time to reach peak plasma acetaminophen concentration and absorption rate constant. A CRI of neostigmine increased fecal production and frequency of urination. Neostigmine induced a dose-dependent increase of contractile amplitude in jejunum and pelvic flexure muscle strips. Incubation of muscle strips with atropine and DAU 5884 inhibited the response to acetylcholine and neostigmine. Incubation of smooth muscle strips from the jejunum with edrophonium increased the response to acetylcholine and had no effect on the response to neostigmine in vitro.

Conclusions and Clinical Relevance—A CRI of neostigmine increased fecal production and urination frequency in horses. A CRI of neostigmine did not decrease gastric emptying. Neostigmine stimulated contractile activity of jejunum and pelvic flexure smooth muscle strips in vitro.

Abstract

Objective—To determine the response to neostigmine of the contractile activity of the jejunum and pelvic flexure and the effects of a continuous rate infusion (CRI) of neostigmine in horses.

Animals—7 adult horses and tissue from 12 adult horses.

Procedures—A CRI of neostigmine (0.008 mg/kg/h) or placebo was administered to 6 horses in a crossover study design. Gastric emptying was evaluated by the acetaminophen test. The frequency of defecation and urination and the consistency and weight of feces were recorded throughout the experiment. The effect of neostigmine on smooth muscle contractile activity was evaluated in tissues from the jejunum and pelvic flexure. The effect of neostigmine and acetylcholine after incubation with muscarinic receptor antagonists (atropine and DAU 5884) and an acetylcholinesterase inhibitor (edrophonium) was also investigated in vitro.

Results—No difference was observed between neostigmine and placebo for time to reach peak plasma acetaminophen concentration and absorption rate constant. A CRI of neostigmine increased fecal production and frequency of urination. Neostigmine induced a dose-dependent increase of contractile amplitude in jejunum and pelvic flexure muscle strips. Incubation of muscle strips with atropine and DAU 5884 inhibited the response to acetylcholine and neostigmine. Incubation of smooth muscle strips from the jejunum with edrophonium increased the response to acetylcholine and had no effect on the response to neostigmine in vitro.

Conclusions and Clinical Relevance—A CRI of neostigmine increased fecal production and urination frequency in horses. A CRI of neostigmine did not decrease gastric emptying. Neostigmine stimulated contractile activity of jejunum and pelvic flexure smooth muscle strips in vitro.

Adynamic ileus is characterized by loss of propulsive contractile activity and gastrointestinal tract coordination, leading to accumulation of fluids and formation of gas in the gastrointestinal tract, causing discomfort.1 The causes of adynamic ileus are multifactorial, and when it develops as a complication of gastrointestinal tract surgery, it is identified as POI.2–7 Reported conditions and factors associated with POI include systemic shock, type and location of lesion, duration of surgery and anesthesia, need and length of resection, and type of anastomosis.2,4–8 Recent studies4,7,9 found an overall prevalence of POI after colic surgery from 14% to 22%. More than 50% of fatalities after colic surgery occur in the postoperative period,10 and the most common reasons for death or euthanasia during the postoperative period are POI, circulatory or endotoxic shock, and persistent signs of pain.11,12

Because of the multifactorial and complex causes of ileus in horses, a variety of treatment options have been proposed, including the use of pharmacological agents to increase propulsive activity of the intestine. Prokinetic agents (cholinomimetics, adrenergic antagonists, benzamides, macrolide antimicrobials, dopamine antagonists, local anesthetics, and phenoxybenzamine) have been used in horses with variable success.13–16 Neostigmine, a parasympathomimetic agent, prolongs the activity of acetylcholine by inhibiting the acetylcholinesterase enzyme, retarding the breakdown of acetylcholine at the synaptic junction.17 Neostigmine has been evaluated in vivo in the proximal and distal portions of the gastrointestinal tract of horses and ponies. Administration of a dose of neostigmine methylsulfate (0.022 mg/kg) repeated 4 times at 30-minute intervals delays gastric emptying in healthy horses.18 A single dose of neostigmine increases the amplitude of rhythmic contractions in isolated jejunal segments of healthy anesthetized ponies19 and increases myoelectrical activity of the ileum20 or has no effect on myoelectrical and mechanical activity of distal jejunum in vivo.21 In the large intestine of healthy ponies, a single dose of neostigmine stimulates propulsive motility in the colon21,22 and improves cecal emptying20 in vivo. Continuous rate infusion of neostigmine promotes defecation in human patients with colonic ileus23; however, it fails to increase gastric emptying and enteral feed absorption in critically ill patients.24 Neither the in vivo effects of a CRI of neostigmine on gastric emptying and defecation frequency in clinically normal horses nor the in vitro effects of neostigmine in the gastrointestinal tract of horses have been evaluated. The objectives of the study reported here were to determine the effects of a CRI of neostigmine on gastric emptying, fecal production, defecation, and urination frequency in healthy horses in vivo and to determine the response to neostigmine of the contractile activity of equine jejunum and pelvic flexure muscle strips in vitro. We hypothesized that neostigmine would accelerate gastric emptying and defecation frequency in healthy horses and that, in vitro, it would stimulate jejunum and pelvic flexure contractility.

Materials and Methods

In vivo study—Seven adult horses (4 females and 3 geldings) from the Center for Equine Health, University of California-Davis, were used in the in vivo part of the study. Horse breeds were Quarter Horse (n = 2), Thoroughbred (1), Standardbred (1), Saddlebred (1), Morgan (1), and Paint (1). Median age and body weight were 17 years (range, 9 to 21 years) and 576 kg (range, 508 to 616 kg), respectively. All procedures were approved by the University of California Institutional Animal Care and Use Committee. The selected horses were healthy and had no recent colic episode or previous abdominal surgery.

One horse was used to determine the time needed for a CRI of neostigmine to reach a steady state. A catheter was aseptically inserted into each jugular vein, one for neostigmine administration and the other for blood collection. Neostigmine methylsulfatea was administered as a CRI by use of a syringe pump to deliver 0.008 mg/kg/h for 6 hours. Venous blood (3 mL) was collected for neostigmine determination from the opposite jugular catheter into heparinized evacuated tubes before infusion (baseline) and during the infusion period at 5, 10, 20, 30, 40, and 50 minutes and at 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and 6 hours. Immediately after collection, blood was centrifuged at 2°C for 5 minutes, and the plasma was collected, immediately frozen at −20°C, and stored at −80°C on the same day.

The other 6 horses were used in a randomized crossover design with a 2-week interval between studies. Before and between studies, horses were kept in a dry (nonirrigated) pasture and were fed a mixture of 50% alfalfa hay and 50% grass hay ad libitum. Horses were moved to a stall 24 hours before the experiment. Horses were fed a mixture of alfalfa hay and grass hay at 1% of body weight twice daily while stabled. Food and water were withheld for 12 and 6 hours, respectively, before initiation of the study. Horses did not receive food or water during the duration of the experiment. A catheter was aseptically placed in each jugular vein. One catheter was used for the CRI of neostigmine or placebo and the other catheter for blood sampling and crystalloid fluid administration. Horses were sedated with 200 mg of xylazine hydrochloride, and a nasogastric tube was placed before the continuous infusion was started. Once the nasogastric tube was in the stomach, it was marked to keep the same length of tube in the stomach throughout each experiment. The nasogastric tube was left in place until the end of each study (5.5 hours). For each experiment, each horse received either a continuous IV infusion of neostigmine methylsulfate (0.008 mg/kg/h) or the same volume of saline (0.9% NaCl) solution (placebo) for 5.5 hours. During the entire experiment, horses received crystalloid fluidsb at a maintenance rate (2 mL/kg/h). Acetaminophenc powder (20 mg/kg, mixed with 400 mL of deionized water) was administered via the nasogastric tube followed by 100 mL of deionized water 3 hours after initiation of neostigmine or placebo infusion. Venous blood (3 mL) was collected into heparinized evacuated tubes for acetaminophen determination immediately before administration (time 0; baseline) and 2, 5, 7, 10, 15, 20, 30, 45, 60, 90, 120, and 150 minutes after acetaminophen administration. Immediately after collection, blood was centrifuged at 2°C for 5 minutes, and the plasma was collected and immediately frozen at −20°C and stored at −80°C on the same day. During the entire experiment, horses were videotaped and closely monitored for signs of abdominal pain. Physical examinations were performed (heart and respiratory rates and rectal temperature) at baseline and during the CRI at 5, 10, 20, 30, 40, and 50 minutes and at 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and 5.5 hours. Frequencies of defecation and urination and consistency of feces were recorded, and the feces were weighed. Fecal consistency was graded by one of the authors (BM) by application of a grading system used in the hospital's intensive care unit, where 1 = normal dry feces, 2 = soft solid feces maintaining a form, 3 = soft nonformed feces, 4 = soft nonformed feces with watery projectile diarrhea, and 5 = only watery projectile diarrhea. The feces from each defecation episode were graded, and the mean from all defecation episodes was used for statistical analysis. After each experiment, the nasogastric tube and IV catheters were removed, and horses were fed and kept overnight in the stall for observation.

Acetaminophen concentration was quantified in plasma from horses via liquid chromatography–tandem mass spectrometry after extraction cleanup with a protein precipitation procedure as described.25 The concentration of acetaminophen in each sample was determined by use of an internal standard method by use of peak area ratio and linear regression analysis. The technique was optimized to provide a minimum limit of quantification of 10 ng/mL for acetaminophen. Determination of plasma neostigmine concentration was performed by high-performance liquid chromatography–tandem mass spectrometry.d After extraction cleanup with a protein precipitation procedure, the concentration of neostigmine in each sample was determined by use of an internal standard (benzyldimethylphenyl ammonium) method by use of peak area ratio and linear regression analysis. The technique was optimized to provide a lower limit of quantification of 10 pg/mL, and the lower limit of detection was 5.0 pg/mL.

Gastric emptying was evaluated by the acetaminophen absorption test.25–29 Pharmacokinetic data analysis was performed with a commercial software program,e and plasma acetaminophen concentration-time data were assessed by use of noncompartmental analysis modeling. Peak plasma acetaminophen concentration and Tmax were estimated from the data. Linear trapezoidal areas were used in calculating the plasma acetaminophen AUC, and other pharmacokinetic parameters were determined by use of standard noncompartmental equations. Individual modeling of plasma acetaminophen concentrations was performed with a 1-compartment model with first-order absorption and first-order elimination. The model was parameterized with absorption lag time, apparent volume of distribution at steady state, Ka, and elimination rate constant.

In vitro study—Tissue was obtained from 12 adult horses with a median age of 13.6 years (range, 8 to 18 years). Tissue from 6 horses was used for dose response testing, and tissue from the other 6 horses was used to determine the effect of neostigmine and acetylcholine after incubation with muscarinic receptor antagonists and an acetylcholinesterase inhibitor. Horse breeds were Quarter Horse (n = 6), Arabian (2), Paint (1), Thoroughbred (1), Warmblood (1), and Lusitano (1). None of the horses had gastrointestinal tract disorders, had evidence of systemic disease, or were receiving any medications. Horses were euthanatized by IV administration of an overdose of pentobarbital sodium, for reasons unrelated to the study. Segments from the antimesenteric border of the pelvic flexure and midportion of the jejunum (4 vascular arcades orad from the ileum) were collected immediately after euthanasia. Ingesta were removed by washing the lumen with KRB, which contained 110mM NaCl, 4.6mM KCl, 2.5mM CaCl2, 24.8mM NaHCO3, 1.2mM KH2PO4, 1.2mM MgSO4, and 5.6mM glucose and had a pH of 7.3 to 7.4 when equilibrated with 95% O2 and 5% CO2. After washing, the tissue was placed in cold oxygenated KRB. All samples were tested the same day of collection as described.25 Briefly, segments of tissue were pinned flat in a dissecting dish containing KRB. Full-thickness muscle strips (2 × 10 mm) were cut parallel to the circular muscle fibers, and the mucosa and submucosa were removed with the aid of a dissecting microscope. The muscle strips were suspended in organ baths containing 20 mL of continuously oxygenated KRB at 37°C. The distal end of each strip was attached to a glass hook tissue support, and the proximal end was attached to an isometric force transducer. Strips were allowed to rest without tension for 30 minutes and then stretched with the tension of 1 g followed by an additional 1 g 20 minutes later, to receive 2 g of stretch. Experiments were performed with the muscle strips under this tension. This degree of muscle tension was determined in a previous study30 to result in maximal active tension development in the equine circular muscle. The same tension was used for the pelvic flexure smooth muscle to maintain a constant tension in all tissues used for the dose-response testing. The KRB was changed every 30 minutes throughout the equilibration period.

Isometric force was recorded by use of force transducers connected by a transducer cable to an 8-channel polygraph chart recorder and a computer. Data were recorded and analyzed by use of a software package.f Data were normalized by subtracting baseline values either before the cumulative dose responses or after incubation with the specific antagonists.

Cumulative dose responses to neostigmine methylsulfateg were evaluated on midjejunum (n = 14 experimental and 14 control strips from 6 horses) and pelvic flexure (14 experimental and 14 control strips from 6 horses) muscle strips. Experimental (neostigmine) and control (vehicle) strips for each horse were run concurrently. After 90 minutes of stabilization, baseline values were recorded for 3 minutes. Cumulative concentrations of neostigmine or vehicle were added (10−9 to 10−4M) every 3 minutes, and the contractile activity was recorded.

To determine the mechanism of action of neostigmine in equine smooth muscle, muscle strips of jejunum were incubated with a nonselective muscarinic receptor antagonist (atropine sulfateh [10−6M] for 20 minutes; n = 14 experimental and 14 control strips from 6 horses), an M3 selective muscarinic receptor antagonist (DAU 5884 hydrochloridei [10−6M] for 20 minutes; n = 12 experimental and 12 control strips from 6 horses), or a reversible acetylcholinesterase inhibitor (edrophonium chloridej [10−4M] for 3 minutes; 12 experimental and 12 control strips from 6 horses). Control strips were incubated with the same volume of vehicle. After incubation, the dose of acetylcholine (1.28 × 10−6M) or neostigmine (2.5 × 10−6M) that induced 50% of the maximum response, determined from preliminary experiments, was added to experimental and control strips, and the contractile activity was recorded for 10 minutes.

DAU 5884 hydrochloride was dissolved in distilled water and diluted in KRB. Acetylcholine chloride, neostigmine, edrophonium, and atropine sulfate were dissolved in KRB. Active contractile force was adjusted for cross-sectional area as described.25

Statistical analysis—To determine whether a CRI of neostigmine had a significant (P < 0.05) effect on acetaminophen pharmacokinetic variables in vivo, the Wilcoxon rank test was used to compare Cmax, Tmax, Ka, and AUC. Frequencies of defecation and urination, mean values of fecal consistency, and amount of feces produced between treatments were also analyzed by the Wilcoxon rank test. Heart rate, respiratory rate, and body temperature were compared by 2-way repeated-measures ANOVA to determine a difference between treatments.

To determine difference between treatments (control vs neostigmine) in vitro, a 2-way repeated-measures ANOVA was performed. To determine whether neostigmine or vehicle had an effect on the contractile activity of muscle strips, compared with baseline values, the amplitudes of the contractions in response to increasing doses were compared by use of a repeated-measures ANOVA followed by the Bonferroni correction.

To determine whether incubation of muscle strips with a selective and nonselective muscarinic receptor antagonist or with a reversible acetylcholinesterase inhibitor had an effect on the response to neostigmine or acetylcholine, comparisons were performed within treatments (compared with baseline values) and between treatments (vehicle or agent) by use of the Wilcoxon rank test.

Data are presented as mean ± SEM or median (range). All comparisons were performed by use of statistical computer software,k and significance was set at P < 0.05.

Results

In vivo study—The plasma concentration resulting from a CRI of neostigmine (0.008 mg/kg/h) in the pilot horse was graphed (Figure 1). Neostigmine was first detected in the 10-minute sample. Neostigmine concentrations reached a steady state after 2.5 hours of infusion; the concentration remained stable for the remaining 3.5 hours of infusion.

Figure 1—
Figure 1—

Plasma neostigmine concentrations before (0 minutes) and during a neostigmine methylsulfate CRI (0.008 mg/kg/h) in a horse. Neostigmine was infused for 360 minutes to determine the time required to obtain a steady-state concentration (arrow).

Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.579

A CRI of neostigmine (0.008 mg/kg/h) was well tolerated by 3 horses (pilot horse and 2 experimental horses). Three horses developed mild signs of colic (pawing) at 1.5, 2, and 2.5 hours of the neostigmine infusion, and the signs lasted for < 15 minutes. The signs of discomfort resolved with no intervention, and the horses appeared comfortable for the rest of the infusion period. One horse developed more severe signs of colic after 5 hours of the neostigmine infusion. The horse was pawing and sweating and tried to lie down. The horse was walked in the stall for 20 minutes. Neostigmine infusion was stopped 10 minutes prior to the scheduled end of the experiment. After the last blood sample was collected (5.5 hours), the horse received an IV dose of flunixin meglumine (1 mg/kg). The colic signs resolved within minutes. Data from the horse were included in the analysis.

No differences for heart and respiratory rates and body temperature were observed between treatments. Median and range values for amount of feces, consistency of feces, and frequencies of defecation and urination were calculated (Table 1). There was no difference for the consistency of feces (P = 0.068) or defecation frequency (P = 0.066) between treatments. The amount of feces passed was higher (P = 0.046) and urination was more frequent (P = 0.039) with the neostigmine than with the control treatment.

Table 1—

Amount and consistency of feces and frequency of defecation and urination in 6 horses that received a CRI of neostigmine (0.008 mg/kg/h) and saline (0.9% NaCl) solution for 5.5 hours in a crossover study design.

GroupFeces (kg)Feces (mean consistency)Defecation (frequency)Urination (frequency)
Control1.5 (1–2.3)1 (0–1)1.5 (0–3)0 (0–1)
Neostigmine2.9 (1.7–8.1)*1.4 (1–2.9)3.5 (1–10)1 (1–15)*

Values are median (range).

Significant (P < 0.05) difference between treatments.

Curve-fitted data for each treatment group were determined (Figure 2), and pharmacokinetic variables of the absorption of acetaminophen for both groups were calculated (Table 2). The pharmacokinetic values for acetaminophen absorption AUC and Cmax were higher with the neostigmine treatment than the control infusion. In 1 horse from the control group, the acetaminophen concentrations did not fit the absorption patterns of the parameter estimation software, and Ka could not be determined.

Figure 2—
Figure 2—

Curve-fitted data of plasma acetaminophen concentration (mean ± SEM values) for 6 horses administered acetaminophen at 20 mg/kg and a CRI of neostigmine (0.008 mg/kg/h; squares) or saline (0.9% NaCl) solution (circles). Acetaminophen was administered after a 3-hour infusion of neostigmine or saline solution.

Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.579

Table 2—

Pharmacokinetic parameters for acetaminophen absorption (20 mg/kg, PO) in 6 horses that received a CRI of neostigmine (0.008 mg/kg/h) and saline solution (control) in a crossover study design.

 ControlNeostigmine 
VariableMedianRangeMedianRangeP value
Tmax (min)2010–602015–600.85
Cmax (ng/mL)19,17812,153–23,83328,26214,274–33,4580.046
AUCall (min•ng/mL)1,9821,634–2,3512,4271,946–2,7180.028
Ka (1/min)0.2030.09–1.30.1640.06–1.150.463

In 1 horse, Ka could not be determined; therefore, data for the control group only include 5 horses.

In vitro study—Neostigmine induced a significant dose-dependent increase from baseline values in contractile amplitude of the midjejunum from 10−6M (mean ± SEM, 31 ± 27 g/cm2) to 10−4M (493 ± 152 g/cm2). Similar significant increases were evident for the pelvic flexure from 10−5M (228 ± 143 g/cm2) to 10−4M (508 ± 175 g/cm2; Figure 3).

Figure 3—
Figure 3—

Mean ± SEM contractile force detected after addition of increasing concentrations of neostigmine (10−9 to 10−4M [squares]) or control solution (circles) to isolated strips of circular smooth muscle obtained from the midportion of the jejunum (A; n = 14 experimental and 14 control strips from 6 horses) and the pelvic flexure (B; 14 experimental and 14 control strips from 6 horses). *Significantly (P < 0.05) different from baseline values.

Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.579

Incubation of muscle strips from the jejunum with atropine sulfate and DAU 5884 hydrochloride inhibited the response to acetylcholine and neostigmine (Figure 4). Incubation of muscle strips from the jejunum with edrophonium chloride increased the response to acetylcholine and had no effect on the response to neostigmine (Figure 5).

Figure 4—
Figure 4—

Mean ± SEM contractile force detected after addition of a half–maximal-effective-concentration dose of neostigmine (2.5 × 10−6M) or acetylcholine (Ach [1.28 × 10−6M]) to isolated muscle strips of circular smooth muscle obtained from the midportion of the jejunum of 6 horses. Strips were incubated for 20 minutes with atropine or vehicle (A) or DAU 5884 hydrochloride or vehicle (B). After neostigmine or acetylcholine administration, the contractile activity was recorded for 10 minutes. n = No. of strips or horses. #Significantly (P < 0.05) different from vehicle group. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.579

Figure 5—
Figure 5—

Mean ± SEM contractile force detected after addition of a half–maximal-effective-concentration dose of neostigmine (2.5 × 10−6M) or acetylcholine (1.28 × 10−6M) to isolated muscle strips of circular smooth muscle obtained from the midportion of the jejunum of 6 horses. Strips were incubated for 3 minutes with edrophonium chloride or vehicle. After neostigmine or acetylcholine administration, the contractile activity was recorded for 10 minutes. See Figures 3 and 4 for remainder of key.

Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.579

Discussion

Despite advances in understanding of intestinal injury and repair, POI remains an important cause of morbidity and death in horses. Because of multifactorial causes, a reduced number of available prokinetic agents, and a limited number of specific studies in horses, the efficacy of prokinetic agents and the guidelines for their use in the perioperative treatment of horses considered at risk of developing ileus are unclear.13 A survey16 on the use of prokinetic agents in horses with gastrointestinal tract injury revealed that neostigmine is the first choice by veterinarians for lesions involving the large intestine. Reported doses of neostigmine for use in horses are 0.02 to 0.04 mg/kg, IV or SC, at intervals determined by the horse's response.31 However, similar doses have induced signs of abdominal discomfort in ponies in an experimental setting.21 In addition, neostigmine administration in humans has been associated with abdominal cramps, diarrhea, salivation, bradycardia, hypotension, and bronchoconstriction.32 To decrease potential adverse effects and to maintain stable plasma concentrations, some agents are used as continuous infusions. A CRI prevents the sudden peaks and valleys associated with intermittent bolus administrations. Medications currently given as CRIs in equine postoperative colic cases include lidocaine and metoclopramide.6,33,35 Because no pharmacokinetic studies of neostigmine in horses have been reported, for the present study, the mean of the CRI dosages reported for human patients (0.008 mg/kg/h) was selected.23,24 To determine whether the 0.008 mg/kg/h dosage was safe to administer to horses and to determine the time required for a CRI of neostigmine to reach stable blood concentration, a pilot horse received a CRI of neostigmine for 6 hours. Plasma neostigmine concentrations were first detected 10 minutes after initiation of the CRI. Stable plasma concentrations of neostigmine were detected by 2.5 hours of infusion, and these concentrations remained stable to the end of infusion (6-hour sample). To obtain steady state concentrations more quickly, the administration of a bolus at the beginning of the infusion or administration of double the infusion rate for the first half-life of the drug is recommended.36 However, considering that signs of abdominal pain may be induced by neostigmine administration, use of a bolus is not recommended.

Neostigmine was tolerated by 6 of the 7 horses that received a CRI of neostigmine. However, mild signs of discomfort were observed for a short period (< 15 minutes) in 3 horses, mainly before passing feces, and the horses had no signs of discomfort for the rest of the infusion without any treatment or discontinuation of the drug. However, more severe signs of pain were observed in 1 horse, for which the infusion needed to be discontinued and the horse consequently treated with analgesics. Administration of neostigmine as in this study should be used with caution in clinical cases because horses at risk of or with POI may already have visceral pain. Agents with a short elimination half-life benefit from a CRI because therapeutic concentrations are easier to maintain. In addition, the rate of infusion can easily be changed to induce the desired effect. After stopping the infusion, the amount or concentration of drug in the body decreases by one-half for each half-life.36 Therefore, discontinuation of the infusion is all that may be needed to stop adverse effects.

Neostigmine increases progressive motility of the large intestine and induces defecation in healthy ponies.21,37 In agreement with these studies, a CRI of neostigmine induced an increase in the amount of feces passed, compared with the control treatment in the present study. Although no significant differences for consistency of feces and defecation frequency were observed, the P value (P = 0.07) was close to 0.05. It would have been interesting to measure fecal water content to determine whether the difference in weight of feces was attributable to an increase in water content or the amount of feces produced. Differences in the amount of food consumed could also be responsible for the differences in weight of feces; however, the feeding protocol was the same for all horses, and each horse was used as its own control to prevent variations.

An adverse effect of neostigmine is increased urinary frequency,l and a current indication for use of acetylcholinesterase inhibitors is atony of the smooth muscle of the urinary bladder.38 Therefore, the stimulation of urination observed in the present study after neostigmine administration is in agreement with the literature.

In the present study, light sedation with xylazine was used 30 minutes before the experiment to facilitate catheter placement and nasogastric intubation and to prevent a difference in management between nervous horses and calm horses. However, we cannot completely rule out the possibility that sedation had some effects on gastrointestinal tract motility. The liquid and solid phase of gastric emptying is affected by xylazine (1 mg/kg).39,40 However, the effect of a lower dose of xylazine (0.5 mg/kg) on solid-phase gastric emptying rate does not differ from that of saline solution administration.39 In addition, xylazine (0.5 mg/kg) induces a mild reduction in duodenal motility that lasts only 30 minutes.41 To decrease adverse effects of sedation, we used a lower dose of xylazine (0.35 mg/kg) and evaluated gastric emptying 3.5 hours after the light sedation. The half-life of elimination of xylazine in horses is 49 minutes, with a body clearance of 21 mL/kg/min.42 Therefore, by 3.5 hours, the effects of xylazine on motility should be minimal.

The acetaminophen absorption test has been used in horses and other species to evaluate the effect of pharmacological agents and nasogastric intubation on gastric emptying of liquids.3,26–29,43–46 Parameters used to evaluate gastric emptying by the acetaminophen absorption test included Cmax, Tmax, AUC, and Ka. However, 2 studies27,43 in horses comparing gastric emptying evaluated by nuclear scintigraphy (considered to be the gold standard) with the acetaminophen absorption test found that only Tmax and Ka correlate with the half-life of liquid-phase gastric emptying. No differences were detected for Tmax and Ka between treatments in the present study, indicating that a CRI of neostigmine neither increased nor decreased gastric emptying of fluids. Studies in horses using the acetaminophen absorption test found that agents known to decrease gastric emptying (atropine and xylazine) had an effect on Cmax or AUC.26,40 A common value used to evaluate gastric emptying by use of by nuclear scintigraphy is the half-life of liquid-phase gastric emptying. However, a recent report45 indicates that such a simple parameter would not be sufficient to compare the curves from different methods and instead that study compared the 3-quartile degrees of retention (75%, 50%, and 25%). In humans, Cmax, Tmax, and AUC from the acetaminophen absorption test have been compared with scintigraphy and evaluated in a systematic literature review.47 Of the 4 studies comparing Cmax with scintigraphy, 2 found satisfactory correlation and 2 found poor correlation. Of the 7 studies comparing AUC with scintigraphy, 3 found good correlation, 2 found moderate correlation, and 2 found poor correlation. Of the 5 studies comparing Tmax with scintigraphy, 3 found good correlation and 2 found poor correlation. If Cmax and AUC are indicators of gastric emptying in horses as has been suggested in other species, then a CRI of neostigmine may be beneficial for the prevention or treatment of horses with POI.

A previous study18 revealed that SC administration of neostigmine (0.022 mg/kg, repeated 4 times every 30 minutes) decreases gastric emptying as evaluated by the passage of markers. However, that study18 used 4 bolus doses over 2 hours; therefore, the total dose was approximately 10 times the dose administered in this study. It is possible that the administration of neostigmine boluses at high doses induces nonpropulsive contractions, causing a delay of gastric emptying, and that lower concentrations over longer time (CRI) may result in a more normal function by maintaining more constant blood concentrations. In addition, differences in the routes of administration (SC vs IV), experimental methodology (transit of plastic markers vs acetaminophen test), materials of gastric emptying evaluated (plastic beads vs liquid), and types of administration (boluses vs CRI) may also be responsible for the differences observed between studies. Because of the reported adverse effects on gastric outflo w, the use of neostigmine has been considered to be contraindicated in most equine postoperative motility disorders.13 The present study found that a CRI of neostigmine did not decrease gastric emptying, as measured by Tmax and Ka, as has been reported.18 Additional studies in live horses evaluating other doses of neostigmine administered as a CRI and the use of a CRI of neostigmine to prevent or treat POI are warranted. It will also be of interest to determine the relevance of finding significant differences in Cmax and AUC when the acetaminophen test is used to evaluate gastric emptying.

An effect of an IV overdose of pentobarbital (for euthanasia) on gastrointestinal tract motility in the present in vitro study cannot be excluded. However, no studies have been performed in horses to determine the effect of different euthanasia methods on smooth muscle contractility. The same method of euthanasia has been used in our laboratory and other institutions in similar equine in vitro studies.25,48–52 Euthanasia by IV pentobarbital overdose in rabbits has no effect on ileal or aortic contractility or on aortic prostaglandin production, compared with decapitation.30 The report of that study30 concluded that pentobarbital overdose is the euthanasia technique that induces the least clinically relevant alterations of vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility, compared with decapitation alone. Therefore, we believe pentobarbital overdose is an acceptable method of euthanasia for these types of studies.

During peristalsis, circular smooth muscle contractions take place in coordination with shortening of the longitudinal muscle, causing transit of ingesta.51 In the present study, only the in vitro effects of neostigmine on the circular smooth muscle layer were evaluated. However, other studies30,51–54 have found different responses between circular and longitudinal smooth muscle layers. Evaluating drug responses of different muscle layers under different stimuli may improve the ability to correlate in vitro results with physiologic and pathological responses.52 Therefore, when possible, evaluation of both circular and longitudinal muscle layers of the intestine is recommended.

Neostigmine administration induced a concentration-dependent increase in the contractile activity of the circular muscle strips of midjejunum and pelvic flexure in the horses of the present study. The contractile response of muscle strips of the midjejunum to half maximal effective concentrations of neostigmine was similar to the response induced by acetylcholine. To try to determine the mechanism of action of neostigmine, jejunal muscle strips were incubated with atropine, a nonselective competitive antagonist for the muscarinic receptor types M1 to M5, and with DAU 5884, a selective muscarinic M3 receptor antagonist. Both neostigmine and acetylcholine failed to elicit a contractile response from jejunal muscle strips incubated with the antagonist. The fact that both antagonists abrogated the effects of neostigmine indicated that neostigmine, either directly or via acetylcholine, acts on the M3 receptor. To determine whether the main mechanism of action of neostigmine was inhibiting the cholinesterase enzyme, muscle strips were incubated with edrophonium chloride, a reversible acetylcholinesterase inhibitor. Incubation of muscle strips with edrophonium increased the response of the smooth muscle to acetylcholine, compared with baseline values and control strips, indicating that the inhibition of acetylcholinesterase increased the availability of acetylcholine. A high concentration (10−4M) of edrophonium was used in the tissue bath in an attempt to completely block acetylcholinesterase activity. By blocking acetylcholinesterase activity, prevention of the contractile response induced by neostigmine was expected. However, muscle strips incubated with edrophonium responded to neostigmine. Edrophonium reduced the contractile response to neostigmine from 391 to 170 g/cm2, compared with control strips; however, the reduction did not reach significance. Similarly, a previous study55 found that incubation of guinea pig ileal muscle strips with dyflos, an irreversible acetylcholinesterase inhibitor, does not affect the contractile response to neostigmine. It is unknown whether edrophonium completely inhibited the acetylcholinesterase in the present study. In addition, edrophonium is a reversible inhibitor, and although a short incubation time (3 minutes) was used, it is not known what percentage of inhibition was still present at the time of neostigmine stimulation. If edrophonium only partially blocked acetylcholinesterase activity, then addition of neostigmine would still have induced an increase in contractile activity similar to that observed in this study. Therefore, additional experiments to determine whether neostigmine has other mechanisms of action in addition to increasing acetylcholinesterase activity or concentration are warranted.

Neostigmine stimulated in vitro contractile activity of the jejunum and pelvic flexure muscle strips. A CRI of neostigmine did not decrease gastric emptying as described elsewhere. A CRI of neostigmine was safe to administer and increased the amount of feces produced and frequency of urination in healthy horses.

ABBREVIATIONS

AUC

Area under the curve

Cmax

Peak plasma acetaminophen concentration

CRI

Continuous rate infusion

Ka

Absorption rate constant

KRB

Modified Krebs Ringer buffer solution

POI

Postoperative ileus

Tmax

Time to reach peak plasma acetaminophen concentration

a.

Neostigmine methylsulfate, Baxter Healthcare Corp, Deerfield, Ill.

b.

Plasma-lyte A, Baxter, Deerfield, Ill.

c.

Acetaminophen, Sigma-Aldrich, St Louis, Mo.

d.

Kenneth L. Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California-Davis, Davis, Calif.

e.

WinNonlin, version 5.0.1, Parsight, Palo Alto, Calif.

f.

Power Lab, AD Instruments Pty Ltd, Colorado Springs, Colo.

g.

Neostigmine methylsulfate, Sigma-Aldrich, St Louis, Mo.

h.

Atropine sulfate, Sigma-Aldrich, St Louis, Mo.

i.

DAU 5884 hydrochloride, Tocris Bioscience, Ellisville, Mo.

j.

Edrophonium chloride, Bioniche Pharma, Lake Forest, Ill.

k.

SPSS, version 10.0.5, SPSS Inc, Chicago, Ill.

l.

Ross MW, Donawick WJ, Sellers AJ, et al. Normal and altered cecocolic motility patterns in ponies (abstr). Vet Surg 1985;14:63.

References

  • 1. Gerring EE, Hunt JM. Pathophysiology of equine postoperative ileus: effect of adrenergic blockade, parasympathetic stimulation and metoclopramide in an experimental model. Equine Vet J 1986; 18:249255.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Blikslager AT, Bowman KF & Levine JF, et al. Evaluation of factors associated with postoperative ileus in horses: 31 cases (1990–1992). J Am Vet Med Assoc 1994; 205:17481752.

    • Search Google Scholar
    • Export Citation
  • 3. Doherty TJ. Postoperative ileus: pathogenesis and treatment. Vet Clin North Am Equine Pract 2009; 25:351362.

  • 4. Cohen ND, Lester GD & Sanchez LC, et al. Evaluation of risk factors associated with development of postoperative ileus in horses. J Am Vet Med Assoc 2004; 225:10701078.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Holcombe SJ, Rodriguez KM & Haupt JL, et al. Prevalence of and risk factors for postoperative ileus after small intestinal surgery in two hundred and thirty-three horses. Vet Surg 2009; 38:368372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Torfs S, Delesalle C & Dewulf J, et al. Risk factors for equine postoperative ileus and effectiveness of prophylactic lidocaine. J Vet Intern Med 2009; 23:606611.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Roussel AJ Jr, Cohen ND & Hooper RN, et al. Risk factors associated with development of postoperative ileus in horses. J Am Vet Med Assoc 2001; 219:7278.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Freeman DE, Hammock P & Baker GJ, et al. Short- and long-term survival and prevalence of postoperative ileus after small intestinal surgery in the horse. Equine Vet J Suppl 2000;(32):4251.

    • Search Google Scholar
    • Export Citation
  • 9. Mair TS, Smith LJ. Survival and complication rates in 300 horses undergoing surgical treatment of colic. Part 2: short-term complications. Equine Vet J 2005; 37:303309.

    • Search Google Scholar
    • Export Citation
  • 10. Hunt JM, Edwards GB, Clarke KW. Incidence, diagnosis and treatment of postoperative complications in colic cases. Equine Vet J 1986; 18:264270.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Hunt JM, Gerring EL. A preliminary study of the effects of metoclopramide on equine gut activity. J Vet Pharmacol Ther 1986; 9:109112.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Mair TS, Smith LJ. Survival and complication rates in 300 horses undergoing surgical treatment of colic. Part 1: short-term survival following a single laparotomy. Equine Vet J 2005; 37:296302.

    • Search Google Scholar
    • Export Citation
  • 13. Dart AJ, Hodgson DR. Role of prokinetic drugs for treatment of postoperative ileus in the horse. Aust Vet J 1998; 76:2531.

  • 14. Koenig J, Cote N. Equine gastrointestinal motility—ileus and pharmacological modification. Can Vet J 2006; 47:551559.

  • 15. Van Hoogmoed LM. Clinical application of prokinetics. Vet Clin North Am Equine Pract 2003; 19:729740.

  • 16. Van Hoogmoed LM, Nieto JE & Snyder JR, et al. Survey of prokinetic use in horses with gastrointestinal injury. Vet Surg 2004; 33:279285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Davis LE, Desmond Baggot J. Gastrointestinal pharmacology. In: Anderson NV, ed. Veterinary gastroenterology. Philadelphia: Lea & Febiger, 1980; 277310.

    • Search Google Scholar
    • Export Citation
  • 18. Adams SB, MacHarg MA. Neostigmine methylsulfate delays gastric emptying of particulate markers in horses. Am J Vet Res 1985; 46:24982499.

    • Search Google Scholar
    • Export Citation
  • 19. Parks AH, Stick JA & Arden WA, et al. Effects of distention and neostigmine on jejunal vascular resistance, oxygen uptake, and intraluminal pressure changes in ponies. Am J Vet Res 1989; 50:5458.

    • Search Google Scholar
    • Export Citation
  • 20. Lester GD, Merritt AM & Neuwirth L, et al. Effect of α2-adrenergic, cholinergic, and nonsteroidal anti-inflammatory drugs on myoelectric activity of ileum, cecum, and right ventral colon and on cecal emptying of radiolabeled markers in clinically normal ponies. Am J Vet Res 1998; 59:320327.

    • Search Google Scholar
    • Export Citation
  • 21. Adams SB, Lamar CH, Masty J. Motility of the distal portion of the jejunum and pelvic flexure in ponies: effects of six drugs. Am J Vet Res 1984; 45:795799.

    • Search Google Scholar
    • Export Citation
  • 22. Rutkowski JA, Ross MW, Cullen K. Effects of xylazine and/or butorphanol or neostigmine on myoelectric activity of the cecum and right ventral colon in female ponies. Am J Vet Res 1989; 50:10961101.

    • Search Google Scholar
    • Export Citation
  • 23. van der Spoel JI, Oudemans-van Straaten HM & Stoutenbeek CP, et al. Neostigmine resolves critical illness-related colonic ileus in intensive care patients with multiple organ failure—a prospective, double-blind, placebo-controlled trial. Intensive Care Med 2001; 27:822827.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Lucey MA, Patil V & Girling K, et al. Does neostigmine increase gastric emptying in the critically ill? Results of a pilot study. Crit Care Resusc 2003; 5:1419.

    • Search Google Scholar
    • Export Citation
  • 25. Maher O, Nieto JE & Stanley SD, et al. Evaluation of the effect of ranitidine on gastroduodenal contractile activity and gastric emptying in horses. Am J Vet Res 2008; 69:11531157.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Doherty TJ, Andrews FM & Provenza MK, et al. Acetaminophen as a marker of gastric emptying in ponies. Equine Vet J 1998; 30:349351.

  • 27. Lohmann KL, Bahr A & Cohen ND, et al. Evaluation of acetaminophen absorption in horses with experimentally induced delayed gastric emptying. Am J Vet Res 2002; 63:170174.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Lammers TW, Roussel AJ & Boothe DM, et al. Effect of an indwelling nasogastric tube on gastric emptying rates of liquids in horses. Am J Vet Res 2005; 66:642645.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Cruz AM, Li R & Kenney DG, et al. Effects of indwelling nasogastric intubation on gastric emptying of a liquid marker in horses. Am J Vet Res 2006; 67:11001104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Rakestraw PC, Snyder JR & Woliner MJ, et al. Involvement of nitric oxide in inhibitory neuromuscular transmission in equine jejunum. Am J Vet Res 1996; 57:12061213.

    • Search Google Scholar
    • Export Citation
  • 31. Fenger CK, Bertone AL, Bertone JJ. Gastrointestinal motility and adynamic ileus. In: Reed SM, Bayly SM, eds. Equine internal medicine. London: WB Saunders Co, 1998; 207215.

    • Search Google Scholar
    • Export Citation
  • 32. Knottenbelt DC. Equine formulary. St Louis: Elsevier, 2006.

  • 33. Kohn CW, Muir WW III. Selected aspects of the clinical pharmacology of visceral analgesics and gut motility modifying drugs in the horse. J Vet Intern Med 1988; 2:8591.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Brianceau P, Chevalier H & Karas A, et al. Intravenous lidocaine and small-intestinal size, abdominal fluid, and outcome after colic surgery in horses. J Vet Intern Med 2002; 16:736741.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Dart AJ, Peauroi JR & Hodgson DR, et al. Efficacy of metoclopramide for treatment of ileus in horses following small intestinal surgery: 70 cases (1989–1992). Aust Vet J 1996; 74:280284.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Tozer TN, Rowland M. Constant-rate regimens. In: Introduction to pharmacokinetics and pharmacodynamics: the quantitative basis of drug therapy. Baltimore: Lippincott Williams & Wilkins, 2006; 169188.

    • Search Google Scholar
    • Export Citation
  • 37. Aquilonius SM, Eckernas SA & Hartvig P, et al. Clinical pharmacology of pyridostigmine and neostigmine in patients with myasthenia gravis. J Neurol Neurosurg Psychiatry 1983; 46:929935.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38. Lacy C, Amstrong L & Goldman M, et al. Drug information handbook. 21st ed. Hudson, Ohio: Lexi-Comp Inc, 2012.

  • 39. Palmer T. Anticholineterase agents. In: Brunton LL, ed. Goodman & Gilman's the pharmacological basis of therapeutics. 12th ed. New York: McGraw Hill, 2011; 239254.

    • Search Google Scholar
    • Export Citation
  • 40. Sutton DG, Preston T & Christley RM, et al. The effects of xylazine, detomidine, acepromazine and butorphanol on equine solid phase gastric emptying rate. Equine Vet J 2002; 34:486492.

    • Search Google Scholar
    • Export Citation
  • 41. Doherty TJ, Andrews FM & Provenza MK, et al. The effect of sedation on gastric emptying of a liquid marker in ponies. Vet Surg 1999; 28:375379.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Merritt AM, Burrow JA, Hartless CS. Effect of xylazine, detomidine, and a combination of xylazine and butorphanol on equine duodenal motility. Am J Vet Res 1998; 59:619623.

    • Search Google Scholar
    • Export Citation
  • 43. Garcia-Villar R, Toutain PL & Alvinerie M, et al. The pharmacokinetics of xylazine hydrochloride: an interspecific study. J Vet Pharmacol Ther 1981; 4:8792.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Lohmann KL, Roussel AJ & Cohen ND, et al. Comparison of nuclear scintigraphy and acetaminophen absorption as a means of studying gastric emptying in horses. Am J Vet Res 2000; 61:310315.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45. Medhus AW, Sandstad O & Bredesen J, et al. Delay of gastric emptying by duodenal intubation: sensitive measurement of gastric emptying by the paracetamol absorption test. Aliment Pharmacol Ther 1999; 13:609620.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Glerup H, Bluhme H & Villadsen GE, et al. Gastric emptying: a comparison of three methods. Scand J Gastroenterol 2007; 42:11821186.

  • 47. Sagara K, Mizuta H & Ohshiko M, et al. Relationship between the phasic period of interdigestive migrating contraction and the systemic bioavailability of acetaminophen in dogs. Pharm Res 1995; 12:594598.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48. Willems M, Quartero AO, Numans ME. How useful is paracetamol absorption as a marker of gastric emptying? A systematic literature study. Dig Dis Sci 2001; 46:22562262.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49. Murray A, Cottrell DF, Woodman MP. Cholinergic activity of intestinal muscle in vitro taken from horses with and without equine grass sickness. Vet Res Commun 1994; 18:199207.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50. Chidambaram RM, Eades SC & Moore RM, et al. Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1. Am J Vet Res 2005; 66:12021208.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51. Lillich JD, Rakestraw PC & Roussel AJ, et al. Expression of the ether-a-go-go (ERG) potassium channel in smooth muscle of the equine gastrointestinal tract and influence on activity of jejunal smooth muscle. Am J Vet Res 2003; 64:267272.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52. Malone ED, Kannan MS & Brown DR, et al. Adrenergic, cholinergic, and nonadrenergic-noncholinergic intrinsic innervation of the jejunum in horses. Am J Vet Res 1999; 60:898904.

    • Search Google Scholar
    • Export Citation
  • 53. Butler MM, Griffey SM & Clubb FJ Jr, et al. The effect of euthanasia technique on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility. Lab Anim Sci 1990; 40:277283.

    • Search Google Scholar
    • Export Citation
  • 54. Nieto JE, Rakestraw PC & Snyder JR, et al. In vitro effects of erythromycin, lidocaine, and metoclopramide on smooth muscle from the pyloric antrum, proximal portion of the duodenum, and middle portion of the jejunum of horses. Am J Vet Res 2000; 61:413419.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55. Cox B, Lomas DM. The effects of eserine and neostigmine on the guinea-pig ileum and on ilial longitudinal muscle strips. J Pharm Pharmacol 1972; 24:541546.

    • Search Google Scholar
    • Export Citation
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