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ANESTHETIC PHARMACOLOGY

Propofol and Midazolam Inhibit Gastric Emptying and Gastrointestinal Transit in Mice

Department of Anesthesiology, Kansai Medical University, Osaka, Japan

Address correspondence and reprint requests to Takefumi Inada, MD, Department of Anesthesiology, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka, 570-8507, Japan. Address e-mail to takefumi{at}wd5.so-net.ne.jp

	Abstract

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We studied the effect of propofol and midazolam on gastric emptying and gastrointestinal transit in mice. Ten minutes after intraperitoneal injection of propofol or midazolam, 0.2 mL of saline containing fluorescent microbeads was infused into the stomach. Thirty minutes later, the gastrointestinal tract was excised, and gastric emptying and gastrointestinal transit were calculated by measuring the quantity of fluorescent microbeads in the gastrointestinal tract by using a flow cytometer. At a dose that produced a light level of sedation (mice righted themselves within 2 s), both drugs significantly, but weakly, inhibited gastric emptying to a similar degree (propofol: P < 0.001 versus control value; 95% confidence interval [CI] for difference, 4.9%–20.2%; midazolam: P < 0.001 versus control value; 95% CI for difference, 7.8%–14.7%). Midazolam, but not propofol, delayed gastrointestinal transit (P < 0.001). At a larger dose that produced a deeper level of sedation (absence of righting reflex >10 s), both drugs significantly inhibited gastric emptying (propofol: P < 0.001; 95% CI for difference, 31.4%–61.2%; midazolam: P < 0.001; 95% CI for difference, 30.8%–61.1%) and gastrointestinal transit (P < 0.001 for both drugs).

IMPLICATIONS: For patients whose lungs are mechanically ventilated, propofol or midazolam may be used to produce sedation. At a light level of sedation, propofol may be preferable to midazolam because of its weaker inhibitory effect on gastrointestinal transit. With an increasing depth of sedation, such an advantage may be reduced.

	Introduction

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In the intensive care unit, it is often necessary to give sedatives, such as propofol and midazolam (1), to alleviate agitation during mechanical ventilation. Those patients may also need nutritional therapy, and enteral feeding has increasingly been used because of its advantages over parenteral nutrition (2,3). Nevertheless, gastric emptying and intestinal transit may often be delayed in critically ill patients (4), and these sedatives may further inhibit motility. The delay may cause intolerance to enteral feeding or overgrowth of bacteria in the gastrointestinal tract and thus may increase the incidence of aspiration pneumonitis (4,5). Therefore, it is preferable to use a sedative that has few inhibitory effects on gastric emptying and gastrointestinal transit.

Subhypnotic and hypnotic doses of propofol in volunteers are reported to have no effect on gastric emptying of liquids (6,7). However, the effects of propofol on intestinal transit are not well known, and little is known about the effects of midazolam on gastric emptying and intestinal transit. In addition, there has been no report comparing the effects of propofol and midazolam on gastric emptying and gastrointestinal transit. Therefore, the aims of our study were to examine whether propofol and midazolam, at sedative doses, inhibit gastrointestinal transit when used to produce comparable levels of sedation.

	Methods

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We used male BALB/c mice (Charles River, Yokohama, Japan) aged 6–7 wk and weighing 23–28 g. The study was approved by the Institutional Committee on Animal Research. The mice were housed under standardized environmental conditions, with a 12-h light/dark cycle. The animals were fasted for 16–24 h but were allowed free access to water until 20–30 min before the start of the experiment. Each animal was individually kept in a wire mesh cage to prevent coprophagy during fasting. All experiments were started between 6:00 and 9:00 AM. These procedures were based on our previous studies (8–10).

We measured gastric emptying and gastrointestinal transit by infusing 0.2 mL of liquid containing fluorescent microbeads into the stomach, and at a fixed time thereafter, measuring the quantity of the microbeads with a flow cytometer (10). We used fluorescent polystyrene microbeads of 6 µm in diameter (Flow Check High Intensity Alignment Grade Particles, 6 µm; Polysciences, Inc., PA) as markers, and we used nonfluorescent microbeads of 0.85 µm in diameter (Sphero purple fluorescent particles; BD Biosciences Pharmingen, CA). The manufacturers provided these microbeads in water: for the 6-µm microbeads, 1 µL of liquid contained 2 x 10³ beads, and for the 0.85 µm microbeads it contained 10⁶ beads.

Each mouse was lightly anesthetized with halothane (2%) by placing the mouse in a clear container until it went to sleep. Then 0.2 mL of saline, containing the 6-µm fluorescent microbeads (50 µL) together with the 0.85-µm nonfluorescent microbeads (1 µL), was infused via a metal cannula (PS 7912, Isis Co., Ltd., Osaka, Japan) into the stomach (10). The smaller nonfluorescent microbeads (0.85 µm) were infused to increase recovery of the 6-µm markers from the gastrointestinal tract (10).

Thirty minutes later, the mouse was killed by an overdose of halothane. The esophagus just proximal to the gastric fundus and the duodenum just distal to the pylorus were cross-clamped, and the stomach and small-intestinal tract were removed. This interval was determined to obtain the geometric center (GC) (see below) of 6–7 and to prevent the leading edge of the test fluid from going beyond the ileocecal junction (10). If there was chyme in either the stomach or small-intestinal tract, data were not used. The intestinal tract was placed on a ruled template and divided into 10 equal segments. The stomach and each segment of intestine were placed into individual tubes containing 5 mL of phosphate-buffered saline. Each tube was then vortexed, and 600 µL of the supernatant (the volume that is often used for measurement with flow cytometry) was filtered through a strainer (Cell-Strainer; BD Biosciences Discovery Labware, MA) and was subjected to flow cytometry.

The quantity of 6-µm fluorescent microbeads in each sample was measured with a flow cytometer (FACScan; BD Biosciences Immunocytometry Systems, CA). The fluorescent 6-µm microbeads are labeled with a fluorescent yellow-green dye, which absorbs the light of a 488-nm argon laser and emits yellow-green fluorescence. In contrast, 0.85-µm microbeads do not emit fluorescence when a 488-nm argon laser is applied. In flow cytometry, the 6-µm microbeads were selected (gated) by their distinct forward scatter (which reflects the size of particles) and by their side light scatter (which reflects the complexity of the inside structure of particles) profiles. The gated particles were further analyzed for the presence of intense fluorescence. The number of particles with this high fluorescence intensity was counted for 30 s with the high flow rate of the cytometer (10).

Gastric emptying of liquids was calculated as

where total count = stomach count + (count in each intestinal segment). Gastrointestinal transit was assessed by using the GC (the center of gravity). This was calculated by a method described previously (8–10) (Fig. 1):

View larger version (16K): [in this window] [in a new window]   Figure 1. Three hypothetical examples of the distribution of markers in the 10 segments of the small intestine. The distribution is expressed as geometric center (the center of gravity) [ (CiSi)/ Ci, where Ci is the count in segment Si) (8–10). A, All the marker is in segment 1; therefore, the geometric center is 1 (1 x 1 + 0 x 2 +...+ 0 x 10 = 1). B, All the marker is in segment 10; therefore, the geometric center is 10 (0 x 1 +...+ 0 x 9 + 1 x 10 = 10). C, The marker is evenly distributed throughout the 10 segments; therefore, the geometric center is 5.5 [0.1 x (1 + 2 + ...+ 10) = 5.5].

There are two ways of expressing the quantification of gastrointestinal transit: the position of the leading edge of the marker and the distribution within the gastrointestinal tract (such as GC). We chose to use the GC, because this method has been shown to be more sensitive than the leading edge method in detecting a possible inhibitory effect (11).

Mice were allocated randomly into propofol and midazolam groups. For each group, 3 mice were grouped as a set, and one of the following 3 different test solutions (in a volume of 10 mL/kg) was injected intraperitoneally for each mouse: for the propofol group, 10% intralipid, propofol 50 mg/kg, or propofol 100 mg/kg; and for the midazolam group, saline, midazolam 25 mg/kg, or midazolam 50 mg/kg. Ten percent intralipid was used to dilute propofol, and saline was used to dilute midazolam. The doses of propofol and midazolam were based on previous studies (12,13), which showed that both large and small doses of these drugs would produce similar levels of depression of consciousness in mice. Ten minutes after the administration of each drug, the test liquid was infused into the stomach, and gastric emptying and gastrointestinal transit were measured. Only sets in which data from all three mice were successfully obtained were used for the study. Experiments were repeated until 8 sets were obtained for both the propofol and midazolam groups.

In different sets of mice (10 mice for each dose of each drug), we assessed whether the doses used produced similar sedative effects. The degree of sedation was determined according to the rating scale of Boast et al. (14), with minor modifications: 0, no obvious impairment and normal righting reflex; +1, wobbling gait during locomotion, but the mouse rights itself within 2 s; +2, latency to righting of >2 s and <10 s; and +3, absence of righting reflex (no reflex within 10 s). The righting reflex was assessed by placing the mouse on its back and observing the time taken to correct its posture. The mean score of righting reflex in triplicate was assessed.

Results are expressed as mean or 95% confidence interval (CI) for the difference. Repeated-measures analysis of variance with Bonferroni correction was used to compare gastric emptying and gastrointestinal transit for each drug at different doses. The Mann-Whitney U-test was used to compare the effects between the two drugs at larger and smaller doses. P < 0.05 was considered statistically significant.

	Results

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The sedation scores were similar between propofol and midazolam both at the small doses and at the large doses (Fig. 2). There was no significant difference in gastric emptying and gastrointestinal transit between mice receiving intraperitoneal intralipid and those receiving intraperitoneal saline (Fig. 3).

View larger version (21K): [in this window] [in a new window]   Figure 2. Depth of sedation after propofol and midazolam. Means and individual data are shown (for clarity, only individual values that are not on the mean values are plotted). Symbols were slightly displaced on the x axis if there were the same values for any given time. Propofol (50 and 100 mg/kg given intraperitoneally [i.p.]) and midazolam (25 mg/kg and 50 mg/kg i.p.) were given to mice (n = 10 for each drug for each experiment), and the depth of sedation was determined (14) as follows: 0, no obvious impairment and normal righting reflex; +1, wobbling gait during locomotion, but the mouse rights itself within 2 s; +2, latency to righting >2 s and <10 s; +3, absence of righting reflex (no reflex within 10 s).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of propofol and midazolam on gastric emptying and gastrointestinal transit. Saline 0.2 mL, containing fluorescent microbeads, was given into the stomach 10 min after intraperitoneal injection. Gastric emptying and gastrointestinal transit were examined 30 min later. Values of the controls are 10% intralipid (for propofol) or saline (for midazolam). Individual values and means are shown. The total count = stomach count + (count in each intestinal segment). *P < 0.001 versus control; #P < 0.05 between propofol and midazolam.

At larger doses, which produced a similar deeper level of sedation, both drugs equally inhibited gastric emptying (propofol: P < 0.001; 95% CI for difference, 31.4%–61.2%; midazolam: P < 0.001; 95% CI for difference, 30.8%–61.1%) and gastrointestinal transit (P < 0.001; 95% CI for difference of GC, 1.5–4.4; midazolam: P < 0.001; 95% CI for difference of GC, 2.3–4.5). There were no significant differences in the inhibitory effect between propofol 100 mg/kg and midazolam 50 mg/kg.

	Discussion

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We have found that, with doses that produced a light level of sedation, both propofol and midazolam significantly inhibited gastric emptying, whereas only midazolam inhibited gastrointestinal transit in mice. At larger doses that produced a deep level of sedation, both propofol and midazolam inhibited gastric emptying and gastrointestinal transit to a similar degree.

There have been several reports of the effects of propofol on gastrointestinal motility. In studies of volunteers, subhypnotic (0.5 µg/mL) and hypnotic (drowsy to arousable on command) doses of IV propofol did not inhibit gastric emptying of liquids (6,7) but slightly delayed orocecal transit (7). In one clinical study, gastric emptying was assessed (by measuring absorption of paracetamol from the small-intestinal tract) after surgery under anesthesia with incremental propofol and nitrous oxide or anesthesia with enflurane and nitrous oxide (15). There was no significant difference between groups. In another clinical study, gastrocecal transit time was determined (by the measurement of end-expiratory hydrogen concentration after infusion of lactulose into the stomach) at the end of surgery (16). There was no significant difference in the transit between anesthesia with propofol and nitrous oxide and anesthesia with isoflurane and nitrous oxide (16). In these two studies, it is not clear whether propofol itself inhibited gastric emptying. In an in vitro study, propofol dose-dependently inhibited gastric and colonic muscle contractions, where 1.2 µg/mL (a sedative dose), but not 0.3 µg/mL (a subhypnotic dose), inhibited acetylcholine-induced gastric and colonic muscle contractions (17). Therefore, it seems that subhypnotic and sedative doses of propofol have a minimum effect on gastric emptying, whereas sedative doses slightly delay intestinal transit.

There have been no reports about the effects of midazolam on gastric emptying. For gastrocecal transit, the transit time was assessed by using the hydrogen exhalation test after elective surgery in one study (18). In that study, there was no significant difference between anesthesia with a continuous infusion of midazolam and ketamine and nitrous oxide and anesthesia with enflurane and nitrous oxide (18). For intestinal motility, subhypnotic doses of IV midazolam (0.03 mg/kg, followed by 0.015 mg/kg 1.5, 3, and 4.5 hours later) had no significant inhibitory effect on the antroduodenojejunal motility in volunteers (19). For the effects of diazepam, another benzodiazepine, IV diazepam 0.2 mg/kg, did not affect gastric emptying in volunteers (20), whereas oral diazepam 0.25 mg/kg increased gastric emptying (21).

Intralipid, the vehicle of propofol, may also affect gastric emptying, because intraduodenal infusion of lipid inhibits gastric emptying (22). Nevertheless, in our study, gastric emptying after intraperitoneal injection of 10% intralipid, in a volume to induce a deep level of sedation by propofol, was similar to gastric emptying after saline. In a previous study of volunteers, 10% intralipid, in a volume equivalent to propofol to achieve a target plasma concentration of 0.5 µg/mL (subhypnotic dose), did not affect gastric emptying (6). However, larger doses of intralipid might affect gastrointestinal motility.

The mechanism of the inhibitory effect of propofol and midazolam on gastric emptying and gastrointestinal transit is not known. One possibility is by activation of the -aminobutyric acid (GABA)_A receptors. GABA_A receptors are found throughout the central nervous system, including the dorsal vagal nuclei, which send cholinergic fibers to the gastrointestinal tract (23). It has been shown that GABA_A receptor-mediated neurotransmission in the dorsal vagal complex enhances the autonomic motility of the small intestine (23). The GABA_A receptors are also found in the enteric nervous system throughout the gastrointestinal tract (24). In the enteric nervous system, GABA_A receptor agonists elicit either excitatory cholinergic or inhibitory nonadrenergic noncholinergic neuron activation, leading respectively to intestinal smooth muscle contraction or relaxation (25,26). However, it is not known whether propofol and midazolam affect the GABA_A receptors involved in gastrointestinal motility.

One limitation of this study was that halothane, which was used to allow insertion of a metal cannula into the stomach, might have delayed gastrointestinal transit (27). Compared with control mice, mice that had been given propofol or midazolam lost consciousness sooner, and thus the exposure of halothane in the study mice would have been less than in the control mice. Therefore, we believe that the possible inhibitory effect of halothane (27) would not have significantly affected the results, because the effect of halothane should be smaller in study mice than in control mice. Another limitation of this study was that propofol and midazolam were given to mice without controlling arterial blood pressure, heart rate, respiration, or body temperature, mainly because of technical difficulties. Therefore, the inhibitory effect of propofol and midazolam on gastric emptying and gastrointestinal transit might not have been a direct effect but could have been the effect of other factors, such as hypotension or depressed respiration. Finally, we examined the degree of sedation by a method described by Boast et al. (14). This simple method has been used by several other researchers, but nonetheless, it may be difficult to differentiate sedation and motor disturbance or unconscious and lethargic states. Therefore, caution is required to seek a drug’s equipotency of sedative effect.

In conclusion, we have shown that, in mice, propofol and midazolam inhibited both gastric emptying and gastrointestinal transit in a dose-dependent manner and that the inhibition was similar with propofol and midazolam at doses that produced a deep level of sedation. In contrast, at small doses that produced a light level of sedation, midazolam, but not propofol, inhibited gastrointestinal transit.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (7) Citing Articles via Google Scholar Google Scholar Articles by Inada, T. Articles by Shingu, K. Search for Related Content PubMed PubMed Citation Articles by Inada, T. Articles by Shingu, K. Related Collections Complications Pharmacology