Epidural analgesia is a mode of pain relief commonly used for labor pain (35%–61% United States1 and 22.5% United Kingdom2). Although the mechanisms responsible for the initiation and progress of labor are still not well understood,3 the effects of epidural analgesia on the progress and outcome of labor have been the focus of a large amount of research in the last decade. Relative to other forms of labor pain relief, epidural analgesia is the most effective but is associated with increased instrumental delivery rate,4–6 longer second stage of labor,4–7 and increased rates of oxytocin augmentation.4–7 One of the mechanisms postulated to account for these epidural analgesia-related adverse effects arises from the direct effects of local anesthetics on the propagation and strength of myometrial contractions.8 Local anesthetics routinely used in obstetric anesthesia practice include bupivacaine and its enantiomer levobupivacaine. Levobupivacaine has a clinical profile closely resembling that of bupivacaine, but is less toxic to both the central nervous and cardiovascular systems.9 These attributes make this drug particularly appealing for use in the setting of obstetric anesthesia, where large volumes of local anesthetic may be required. In vitro findings have suggested that these drugs demonstrate differential effects in their ability to block cardiac sodium and potassium channels,10 which raises the possibility of differential effects in myometrial tissue. Previous studies have reported that bupivacaine11–13 and levobupivacaine13 cause concentration-dependent inhibition of the amplitude of gravid rat myometrial contractions. The effects of bupivacaine and levobupivacaine have not been reported in human myometrium in the in vitro setting. We hypothesized that bupivacaine and levobupivacaine may have differential effects on the amplitude and frequency of human myometrial contractility.
Tissue Sample Preparation
The protocol for the study was approved by the local IRB. After obtaining written informed consent, 10 term ASA physical status 1 pregnant women scheduled for elective lower segment cesarean delivery were enrolled. None of the women were in labor. All patients received spinal anesthesia with 10–12 mg 0.5% hyperbaric bupivacaine, with 20–25 μg of intrathecal fentanyl, and 100–150 μg of intrathecal morphine. After delivery, all women received 5 IU of oxytocin IV (Novartis Pharmaceuticals UK Ltd., Horsham, West Sussex, UK) After the placenta was delivered, a small segment of myometrium was excised from the upper incisional surface of the lower uterine segment. The specimen was washed and stored in lactated Ringer's solution, and refrigerated at 4°C until the experiment began 2–18 h later.
Each specimen was dissected into at least four longitudinal muscle strips 12 mm long, 5 mm wide, and 1 mm thick. The strips were mounted vertically into individual 10- or 15-mL organ baths (Myobath, World Precision Instruments Inc., Sarasota, FL) with Krebs solution and connected to individual force transducers (Transbridge 4M, World Precision Instruments Inc.) to record isometric tension. The Krebs– Henseleit bicarbonate buffer solution contained: NaCl 118 mmol/L, d-glucose 11.1 mmol/L, NaHCO3 24.9 mmol/L, MgSO4 1.2 mmol/L, KCl 4.7 mmol/L, KH2PO4 1.2 mmol/L, and CaCl2 2.5 mmol/L. The organ baths were aerated with a gas mixture of 95% oxygen and 5% CO2 and maintained at 36.7°C. A resting tension of 1g (9.81 mN) (based on protocols from similar experimental work11,12) was initially applied, and subsequently reapplied as necessary over the first 30–40 min until a steady tension was achieved. Simultaneously, the Krebs solution was changed every 10 min for 30 min to remove any residual oxytocin. At no time were contractions stimulated with oxytocin or other uterotonic drugs. Spontaneous contractions usually developed over the following 60–90 min. When the contractions became regular in amplitude and frequency, and after a 30-min control period, the muscle strips were randomly exposed to either bupivacaine (Astrazeneca, UK) or levobupivacaine (Abbott Laboratories, USA). Drug-containing solutions were prepared immediately before each experiment using Krebs solution. The strips were exposed to cumulative concentrations of 10−8 to 3 × 10−4 M for each local anesthetic. There was a 30-min interval between each addition and drugs were added in the following increments: 1 × 10−8 M, 1 × 10−7 M, 3 × 10−7 M, 1 × 10−6 M, 3 × 10−6 M, 1 × 10−5 M, 3 × 10−5 M, 1 × 10−4 M, and 3 × 10−4 M. A control muscle strip was maintained for each sample to which no drugs were added to ensure viability for the duration of the experiment. After the addition of the 3 × 10−4 M concentration of each local anesthetic, the baths were washed out every 10 min with warmed Krebs solution for a period of 30 min to ascertain if the effect of the local anesthetics could be reversed. After completion of the experiment, the weight (0.089 ± 0.01 g) and dimensions of each muscle strip were recorded.
The amplitude (peak force) and frequency of contractions were recorded for each drug treatment window and subsequently analyzed using Powerlab Program (AD Instruments Pty Ltd., Australia). The amplitude of contraction is expressed as a % of the amplitude measured in the control period before the addition of drug. The frequency of contractions was assessed by measuring the peak-to-peak interval, in seconds, between the last two contraction peaks for each drug window and expressed as a percent of the frequency measured in the control period. The amplitude and frequency in the control period were taken as 100%.
Data are presented as mean ± sd. Statistical analysis was performed using GraphPad Prism 4 statistical software (GraphPad Software Inc., USA). Dose–response curves and log EC50 values were generated using nonlinear regression analysis. The effects of levobupivacaine and bupivicaine on contractility were compared using one-way repeated measures analysis of variance. A two-tailed paired t-test was used to compare the effects of increasing bupivacaine and levobupivacaine concentrations to the internal control period. P < 0.05 was considered to be statistically significant.
Eight of the 10 control strips of muscle (to which no drugs were added) contracted without fade for the duration of the experiment (6–7 h). The other two failed to contract and so myometrial strips from these women were excluded. Figure 1 depicts a typical tracing generated by the spontaneous contractions of an isolated myometrial strip, showing a reduction in the amplitude and interval between contractions with increasing concentrations of bupivacaine, and reversibility after drug washout at the end. The mean tension that developed during the control period (n = 8, where n = the number of patients) was 8.61 ± 4.71 g, with a mean peak-to-peak interval of 627 ± 65 s and a contraction rate of 6.9 ± 0.1 contractions per hour.
Local anesthetic effects: There were eight strips of muscle in each of the bupivacaine, levobupivacaine, and control groups. Bupivacaine and levobupivacaine caused a concentration-dependent decrease in the amplitude of contractions (Fig. 2) reaching statistical significance at 1 × 10−4 M (P = 0.002 for levobupivacaine and P = 0.01 for bupivacaine) compared with the control period before the addition of any drugs. This decrease in amplitude of contractions was accompanied by a decrease in peak-to-peak interval, indicating an increased frequency of contractions (Fig. 3). This decrease in peak-to-peak interval differed significantly from the control period at 3 × 10−5 M for both drugs (P = 0.02 for bupivacaine and P = 0.02 for levobupivacaine). There were no significant differences between bupivacaine and levobupivacaine in their effects on myometrial contractility. Table 1 shows the EC50 values (effective concentration which causes 50% of the maximal effect) for amplitude of contractions and interval between contractions for bupivacaine and levobupivacaine. There were no differences between the log EC50 values for these two drugs. The effects of both drugs on contractility were reversible.
Epidural analgesia remains the “gold standard” for providing optimal pain relief in labor. However, its use is associated with increased instrumental delivery rates.4–6 Techniques that use low compared with higher concentration local anesthetic solutions can reduce this rate.14 This implies that local anesthetics may be at least partly responsible for this unwanted effect.
There have been many reasons postulated to explain this effect including (1) the direct effect of local anesthetics on myometrial contractility, (2) the motor block, which may follow epidural analgesia leading to decreased voluntary maternal expulsive efforts during the second stage of labor, (3) paralysis of the pelvic floor muscles that may retard or eliminate the descent and rotation of the fetal head, and (4) attenuation of the normal increase in oxytocin through inhibition of the Ferguson (uteropituitary) reflex.15 We sought to determine if two commonly used local anesthetics in obstetric anesthesia had any direct effect on myometrial contractility in the in vitro setting. We found that both bupivacaine and levobupivacaine caused a concentration-dependent decrease in the amplitude of contractions of term human myometrium. Both drugs caused an increase in frequency of contractions.
The findings of the effects of bupivacaine on amplitude are in accord with previous studies of rat myometrium.11–13 In contrast, Li et al. examined the in vitro effects of levobupivacaine on rat myometrial contractility13 and observed a biphasic effect with a significant increase in amplitude of contractions from 3 × 10−7 M to 3 × 10−6 M, followed by a significant reduction in amplitude of contractions at 1 × 10−4 M. Our findings differ from these in that we observed no increase in contractile amplitude. Many other studies have demonstrated that bupivacaine has an inhibitory effect on contraction in other types of smooth muscle, e.g., canine papillary muscle,16 bladder smooth muscle,17 isolated arterial smooth muscle,18 and tracheal smooth muscle.19
The results of studies of the in vitro effect of local anesthetics on the frequency of myometrial contractions are conflicting. Li et al.13 demonstrated bupivacaine had a biphasic effect increasing contractile frequency at 3 × 10−5 M, whereas both bupivacaine and levobupivacaine showed inhibitory, though nonstatistically significant, effects at 1 × 10−4 M concentration. Other studies have demonstrated conflicting results (an increased contractile frequency12 or no effect on frequency11) on the effects of local anesthetics on frequency of contractions. These differences may be explained by the differences in human and animal myometrium.20 The increase in frequency observed in the current study may be related to altered intracellular calcium content resulting from the reduction in contractile amplitude. In contrast to findings in myocardial tissue, there was no difference observed between bupivacaine and levobupivacaine in their ability to reduce myometrial contractility.10 Local anesthetics are amphiphilic, and can enter a variety of cell compartments and potentially interact with many different cell membranes, organelles (including inhibition of mitochondrial adenosine triphosphate production21), and a variety of both membrane-bound and cytosolic charged molecules. Other mechanisms, which may account for the myometrial inhibitory effect, include blocking of ionotropic signaling pathways (sodium, potassium, or calcium) and interference with G-protein modulation of calcium or potassium channels.22
Do the inhibitory effects of these local anesthetics account for some of the unwanted side effects of labor epidural analgesia? There is only one published study23 reporting plasma levels of bupivacaine and levobupivacaine in parturients after epidural administration. In this study, women received a bolus of 30 mL of 0.5% levobupivacaine or bupivacaine for cesarean delivery anesthesia (150 mg). The maximal plasma concentrations were 1.053 μg/mL (3.24 × 10−6 M) for bupivacaine and 1.017 μg/mL (3.13 × 10−6 M) for levobupivacaine.23 We observed significant inhibitory effects of bupivacaine and levobupivacaine on contractile amplitude at concentrations which were 33 times higher (1 × 10−4 M) than this and 17 times higher than the level at which central nervous system toxicity has been reported to occur (8.07 × 10−6 M for levobupivacaine and 6.92 × 10−6 M for bupivacaine24). The current practice of using low concentration (0.125%–0.25%) solutions of local anesthetics makes it unlikely that the inhibitory plasma concentration of these drug levels would be reached in clinical practice.
This study is limited by the same “experimental noise” that is encountered in all studies of human myometrial contractility; that is, that the myometrial samples were obtained from women undergoing elective cesarean delivery at term. It is not known whether the myometrium has undergone the complex molecular changes required for the onset and establishment of labor at this stage.20 Samples obtained were from the upper incisional surface of the lower uterine segment of the uterus, the only tissue available in this setting. However, regional differences to drugs have been demonstrated in both animal25 and nonpregnant human myometrium,26 and it is possible that different effects may be observed in fundal myometrium. To ensure tissue viability for the duration of the experiment, we limited the exposure of the specimen to each concentration of local anesthetic for 30 min (similar to the protocol used in previously published similar work11). We cannot be certain that the maximal effects of that drug concentration can be observed in that period. In addition, the interaction between the local anesthetics and oxytocin that would occur in normal labor was not examined. Despite three washouts with Krebs solution, we also cannot be certain that some residual oxytocin remained in the myometrial strips. Despite these difficulties, it is important to understand that there are differences between human and animal myometrium, and studies such as this improve our understanding of human myometrial function.
In summary, bupivacaine and its enantiomer levobupivacaine, cause a similar concentration-dependent decrease in human myometrial contractile amplitude in the in vitro setting. This is accompanied by an increase in the frequency of contractions. The concentrations required for the inhibitory effect on the amplitude of contractions is much higher (33 fold) than the clinically relevant plasma concentrations of these drugs after epidural administration and are unlikely to be significant in the setting of low-dose epidural analgesia in labor.
Therefore, a direct effect of local anesthetics on uterine contractility is unlikely to explain the association between epidural labor analgesia and instrumental vaginal delivery.
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