Acta Physiologica Hungarica, Volume 101 (2), pp. 228–235 (2014) DOI: 10.1556/APhysiol.101.2014.2.11
Effects of local anesthetics on contractions of pregnant and non-pregnant rat myometrium in vitro Jin-Song Wei1, Zhe-Bin Jin2, Zhi-Qiang Yin2, Qiang-Min Xie3, Ji-Qiang Chen3, Zi-Gang Li2, Hui-Fang Tang3 1 Department of Medicine, Chinese Medicine Hospital of Jingshan County, Hubei, Jinshan, China Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University, Zhejiang, Hangzhou, China 3 Zhejiang Respiratory Drugs Research Laboratory of the SFDA of China, School of Medicine, Zhejiang University, Hangzhou, China 2
Received: November 20, 2012 Accepted after revision: July 8, 2013 In order to determine whether local anesthetics directly affect the propagation and strength of myometrial contractions, we compared the effects of bupivacaine, ropivacaine, lidocaine and tetracaine on the contractions of myometrium isolated from pregnant and non-pregnant rats. Full-thickness myometrial strips were obtained from 18- to 21-day pregnant and non-pregnant Sprague-Dawley rats and incubated in an organ bath. When spontaneous contractions became regular, strips were exposed to cumulative concentrations of the four local anesthetics ranging from 0.01 to 300 μmol/L and the amplitude and frequency of contraction were recorded. All four compounds caused a concentration-dependent inhibition of the contractility of pregnant and non-pregnant uterine muscle. In pregnant myometrium, the concentration that caused 50% inhibition (IC50) was 100 μmol/L for bupivacaine, 157 μmol/L for ropivacaine, > 1000 μmol/L for lidocaine, and 26.3 μmol/L for tetracaine. In non-pregnant myometrium, the IC50 was 26.9 μmol/L for bupivacaine, 40 μmol/L for ropivacaine, 384 μmol/L for lidocaine, and 7.4 μmol/L for tetracaine. These results suggested that local anesthetics do inhibit myometrial contractions in pregnant and non-pregnant rats in a concentration-dependent manner. Keywords: local anesthetics, bupivacaine, ropivacaine, lidocaine, tetracaine, pregnant, non-pregnant, myometrium, rat, in vitro
The goal of obstetric analgesia is to provide optimal pain relief for the parturient with minimal risk to herself and the fetus. Local anesthetic-induced neural blockade is the most effective means of reducing pain during labor and the postoperative period, and epidural analgesia is in widespread use for gynecologic and obstetric procedures. Compared with other forms of labor pain relief, epidural analgesia is the most effective but increases the risk of instrumental delivery (1, 16). Three of the local anesthetics, bupivacaine, ropivacaine and lidocaine are commonly used in obstetric analgesia, but tetracaine is used only as an adjunct in obstetric patients. Whether epidural analgesia with these anesthetics affects uterine contractility, directly or indirectly, is unclear. Determining the effects of local anesthetics on pregnant myometrium might be one way to assess their therapeutic value for preterm labor, while their effects on non-pregnant myometrium might address their therapeutic value for dysmenorrhea and gynecologic
Corresponding authors: Zi-Gang Li Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University Hangzhou, Zhejiang 310006, China Phone: 86-571-88208230; Fax: 86-571-88208231; E-mail: [email protected] 0231–424X/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
Depressant effects of local anesthetics on rat myometrium
229
surgery. However, to date, comparisons of the in vitro effects of local anesthetic agents on pregnant and non-pregnant myometrium are not well-documented. Previous in vitro studies have suggested that commonly-used local anesthetics alter the spontaneous contractility of pregnant uterine muscle, however, the results are inconsistent. Karsli et al. (9) suggested that bupivacaine at 300 μmol/L decreases the amplitude of contractions, but the frequency increases. Fanning et al. (5) reported similar results in human pregnant myometrium. On the contrary, Arici et al. (2) reported that bupivacaine had no effect on the frequency of uterine contractions, but inhibits rat myometrial contractility significantly more than ropivacaine at 10−4 mol/L. In this study, we evaluated the effects of four commonly-used local anesthetic agents, bupivacaine, ropivacaine, lidocaine and tetracaine (an ester anesthetic) on the contractions of myometrium isolated from pregnant and non-pregnant rats. Materials and Methods Animals Two-month-old, pregnant female Sprague-Dawley rats (n = 8) weighing 270–300 g at 18–20 days of gestation, and 2-month-old, non-pregnant female Sprague-Dawley rats (n = 10) weighing 200 ± 20 g were used. The latter were pretreated with 40 mg diethylstilbestrol subcutaneously for 2 days to induce estrus and improve the response to the drugs. The animals were cared for in accordance with the Guide for the Care and Use of Experimental Animals and the procedures were approved by the Ethics Committee of Zhejiang University School of Medicine. Experimental procedures Pregnant and non-pregnant rats were used for each local anesthetic. After anesthesia with urethane (25%, 4 ml/kg, intravenously), they were killed by cervical subluxation. The preparation of pregnant myometrial strips was performed according to the method described previously (12). The uterine horns were rapidly excised and carefully cleaned from the surrounding connective tissue and opened longitudinally along the mesenteric border. The fetuses of the late-stage pregnant rats were removed and non-uterine tissues were dissected away and discarded. Eight longitudinal full-thickness myometrial muscle strips (measuring 4 × 10 mm) were obtained from each pregnant rat. The uterus of the non-pregnant SpragueDawley rats was rapidly isolated, carefully cleaned of surrounding connective tissue, and 4 strips (10-mm long) were prepared. Eight pregnant and non-pregnant myometrial strips were studied for each local anesthetic. Each strip was mounted vertically, one end connected to the lower hook of the bath and the other to a force rod. The strips were incubated in a 10-ml tissue bath containing DeJalon’s solution (in mM/L: NaCl 153.9, KCl 5.6, glucose 2.7, NaHCO3 5.9, and CaCl2 0.27), which was aerated continuously with 95% O2 and 5% CO2. The pH was maintained at 7.4, and the temperature at 32 ± 0.5 °C to maintain the condition of the strips. The solution was made-up daily for each experiment. The pregnant myometrial strips were allowed to equilibrate at 1 g tension for 20 min until the contractions became regular, as previously reported (12). The characteristics of the contraction frequency and amplitude were recorded by a force displacement transducer (JZ100, Xinhang Machine and Equipment, Gaobeidian, China) coupled to a MedLab Biological Signal Collection System (Medease Science and Technology, Nanjing, China). When the contractions became regular, their frequency and amplitude were recorded as baseline activity, and the local anesthetics were added. Acta Physiologica Hungarica 101, 2014
230
Wei JS et al.
During epidural anesthesia or nerve block, the usual plasma concentrations of the anesthetics are < 100 μmol/L (3, 10, 18). Therefore, we used similar concentrations to compare the effects of the four local anesthetics on the contractility of pregnant and nonpregnant rat myometrium in vitro. Bupivacaine (Shanghai Harvest Pharmaceutical Co. Ltd, No. 4H04009), ropivacaine (Naropin, Astrazeneca AB, No. EF1026), lidocaine (Shanghai Fosun Zhaohui Pharmaceutical Co. Ltd, No. 050308), or tetracaine (Zhejiang Jiuxu Pharmaceutical Co. Ltd, No: 20050405) was added at cumulative concentrations from 0.01 μmol·L–1 to 300 μmol·L–1. Drug-containing solutions were prepared immediately before each experiment. The non-pregnant myometrial strips were allowed to equilibrate at 2 g tension for 40 min, after which contraction was stimulated by oxytocin (0.05 U/10 ml) as previously reported (12). When the contractions became regular, their frequency and amplitude were recorded as baseline activity. The drugs were then added and contraction frequency and amplitude were recorded as described above. The duration of exposure to each concentration was 15 min. The investigators were blinded as to the local anesthetic being used. Each strip was exposed to only one anesthetic agent. Control strips (without drug treatment) were recorded for the same time, and no significant decrement in contraction was observed. The primary end-point of the study was cessation of contractions. Statistical analysis All data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., USA). The concentration that caused 50% inhibition of contraction amplitude (IC50) was calculated by the LD50 data treatment sequence (Blue Cosmos Studio, China). P < 0.05 was considered statistically significant. A)
B)
Fig. 1. Effects of four local anesthetics on the amplitude (A) and frequency (B) of contractions of myometrial strips isolated from non-pregnant rats. Data (mean ± SEM) are expressed relative to baseline values; n = 8; *P < 0.05, **P < 0.01 vs. baseline; +: no contraction Acta Physiologica Hungarica 101, 2014
Depressant effects of local anesthetics on rat myometrium
231
A)
B)
Fig. 2. Effects of four local anesthetics on the amplitude (A) and frequency (B) of contractions of myometrial strips isolated from pregnant rats. Data (mean ± SEM) are expressed relative to baseline values; n = 8; *P < 0.05, **P < 0.01 vs. baseline; +: no contraction
Results In general, all the local anesthetics decreased the amplitude of contraction at the highest concentrations in both the pregnant and non-pregnant uterus (Figs 1A and 2A). The same was true for the frequency of contraction, except for the cases of bupivacaine and lidocaine in the pregnant uterus, which had no significant effect (Figs 1B and 2B). Bupivacaine effects From 1 to 30 μmol/L (pregnant strips), or 1 to 10 μmol/L (non-pregnant strips), bupivacaine did not significantly change the contractile amplitude. However, at 100 μmol/L, it depressed the amplitude in both non-pregnant and pregnant myometrium. In terms of frequency, there was a non-significant tendency to increase up to 30 μmol/L in pregnant myometrial strips (Fig. 2B), but the tendency was to decrease, with significant inhibition at 100 μmol/L, in the non-pregnant strips. Ropivacaine effects Ropivacaine significantly inhibited the amplitude of pregnant and non-pregnant myometrial contractions in a concentration-dependent manner. In pregnant strips, the range was from 1 to 100 μmol/L, and contractility almost ceased at 300 μmol/L. In non-pregnant strips, the range was from 10 to 100 μmol/L and contractions ceased completely at 300 μmol/L. However, ropivacaine tended to slightly increase the frequency at 100 μmol/L in pregnant strips, and at 0.1 μmol/L in non-pregnant strips, but the values did not significantly differ from baseline. Acta Physiologica Hungarica 101, 2014
Wei JS et al.
232
Lidocaine effects Exposure to lidocaine at 100–300 μmol/L significantly decreased the contraction amplitude in both strips. In pregnant strips, it had no effect on contraction frequency even at 300 μmol/L, but the frequency significantly decreased in the non-pregnant strips. Tetracaine effects Tetracaine caused a concentration-dependent inhibition of contractility in pregnant and nonpregnant uterine muscle. It depressed the amplitude of contractions from 30 to 100 μmol/L in pregnant strips, and from 1 to 100 μmol/L in non-pregnant strips (300 μmol/L abolished contractions). In pregnant strips, the response concentration was from 3 μmol/L, and contractions almost ceased at 100 μmol/L. In non-pregnant strips, the frequency was depressed only at 100 μmol/L. Comparison of local anesthetics Based on the above results, tetracaine had a significantly greater depressant effect than the other agents, whereas lidocaine had a significantly lower depressant effect than the others. In terms of IC50, the order of depressant potency in non-pregnant and pregnant myometrium was: tetracaine > bupivacaine > ropivacaine > lidocaine. Based on the IC50 values, the approximate relative local anesthetic potencies of equipotent doses of lidocaine, bupivacaine, ropivacaine and tetracaine were 60:6:4:1 (Table I). Table I. IC50 values for the four local anesthetics on contraction amplitude of pregnant and non-pregnant myometrial strips
IC50
Pregnant (μmol/L)
Non-pregnant (μmol/L)
Bupivacaine
100
26.9
Lidocaine
1771
384
Ropivacaine
157
40
Tetracaine
26.3
7.4
Discussion Bupivacaine is a long-acting local anesthetic of the amide type recommended for infiltration, peripheral nerve block, and epidural/spinal anesthesia. Ropivacaine is a long-acting, amidetype local anesthetic. Its structure and pharmacokinetics are similar to those of bupivacaine; however, ropivacaine has a better cardiotoxicity profile. Ropivacaine is also less lipid-soluble and clears via the liver more rapidly than bupivacaine. Due to its superior safety profile and significantly improved sensory-motor differentiation (11), ropivacaine is currently the longacting anesthetic of choice in our practice. Lidocaine, a short-acting, amino-amide local anesthetic, is not the most widely used in obstetric anesthesia in the USA, but it is the most widely used in China, due to its inherent potency, rapid onset, tissue penetration, effectiveness and cost-effectiveness. Tetracaine is an ester-type local anesthetic with an intermediate to long-duration mode of action. It is mainly used topically in ophthalmology and as an antipruritic, and it has been used in spinal anesthesia. All these local anesthetic agents have been used in obstetric anesthesia and analgesia. Acta Physiologica Hungarica 101, 2014
Depressant effects of local anesthetics on rat myometrium
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Previous studies suggested that bupivacaine causes a concentration-dependent decrease in the amplitude of contractions in pregnant and non-pregnant rat myometrium (2, 12, 14). Studies of human pregnant myometrium in vitro also gave similar results (5, 19). However, some in vivo clinical studies have suggested that bupivacaine has no effect on uterine contractility (15, 17). Furthermore, controversy exists regarding the issue of frequency. Arici et al. (2) reported that bupivacaine has no effect on the frequency of uterine contractions in pregnant 18- to 21-day-old Wistar rats. Karsli et al. (9) indicated that bupivacaine at 300 μmol/L increases the frequency of uterine contractions in albino rats pregnant for 18–21 days. Here, we found that bupivacaine inhibited the amplitude, and tended to increase the frequency in a concentration-dependent manner in pregnant myometrium, consistent with the Fanning et al. report (5), but at higher concentration (100 μmol/L) it had a slightly depressant effect. But in non-pregnant rat myometrium, bupivacaine decreased rather than increased the frequency, suggesting that non-pregnant myometrium may be more sensitive than pregnant myometrium. Our results suggested that ropivacaine had an effect similar to bupivacaine in pregnant and non-pregnant myometrium. Bupivacaine had a greater inhibitory effect on contractility than ropivacaine at higher concentrations (100 and 300 μmol/L), while ropivacaine had a greater inhibitory effect than bupivacaine at lower concentrations (1–30 μmol/L). Arici’s report also showed some similarities with our results (2). In terms of frequency, non-pregnant rat myometrium was more sensitive than pregnant myometrium at 300 μmol/L ropivacaine which completely inhibited the contraction of non-pregnant rat myometrium. A previous study suggested that lidocaine has a significantly greater depressant effect on spontaneous myometrial contractions in the human during late pregnancy than bupivacaine (19), but our results are in contrast to this finding. Based on the IC50, bupivacaine was 17-fold more potent than lidocaine. In pregnant rat myometrium, Fauza et al. demonstrated that lidocaine and ropivacaine both increased the contraction frequency at low concentrations along with a depression in amplitude (6, 7). In our study, lidocaine had no effect on the frequency. In non-pregnant rat myometrium, the results on amplitude were similar to those in pregnant rat myometrium, but on frequency, non-pregnant myometrium was more sensitive than pregnant myometrium at 300 μmol/L. Tetracaine has a recognized use during labor and delivery, but here are no reports recording the effects of the drug on duration of labor, incidence of forceps delivery, status of the newborn, and later growth and development of the child, as well as on the amplitude and frequency in pregnant or non-pregnant myometrium. Maruta and Osa (13) reported that tetracaine inhibited Ca2+ influx and K+ conductance in the uterine longitudinal muscle of estrogen-treated rats. Györke et al. (8) described tetracaine at low concentrations caused an initial inhibition of spontaneous Ca2+-release events in rat ventricular myocytes, while at high concentrations, it blocked release completely. In our study, tetracaine clearly decreased the amplitude and frequency in pregnant and non-pregnant myometrium. It had the highest potency among the four local anesthetics, and pregnant myometrium was more sensitive than non-pregnant myometrium in frequency, but non-pregnant myometrium was more sensitive than pregnant myometrium in amplitude. This suggested that tetracaine may be harmful in pregnancy. In the clinic, the concentrations of local anesthetics achieved in the human myometrium with normal administration in the epidural space are
Effects of local anesthetics on contractions of pregnant and non-pregnant rat myometrium in vitro Jin-Song Wei1, Zhe-Bin Jin2, Zhi-Qiang Yin2, Qiang-Min Xie3, Ji-Qiang Chen3, Zi-Gang Li2, Hui-Fang Tang3 1 Department of Medicine, Chinese Medicine Hospital of Jingshan County, Hubei, Jinshan, China Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University, Zhejiang, Hangzhou, China 3 Zhejiang Respiratory Drugs Research Laboratory of the SFDA of China, School of Medicine, Zhejiang University, Hangzhou, China 2
Received: November 20, 2012 Accepted after revision: July 8, 2013 In order to determine whether local anesthetics directly affect the propagation and strength of myometrial contractions, we compared the effects of bupivacaine, ropivacaine, lidocaine and tetracaine on the contractions of myometrium isolated from pregnant and non-pregnant rats. Full-thickness myometrial strips were obtained from 18- to 21-day pregnant and non-pregnant Sprague-Dawley rats and incubated in an organ bath. When spontaneous contractions became regular, strips were exposed to cumulative concentrations of the four local anesthetics ranging from 0.01 to 300 μmol/L and the amplitude and frequency of contraction were recorded. All four compounds caused a concentration-dependent inhibition of the contractility of pregnant and non-pregnant uterine muscle. In pregnant myometrium, the concentration that caused 50% inhibition (IC50) was 100 μmol/L for bupivacaine, 157 μmol/L for ropivacaine, > 1000 μmol/L for lidocaine, and 26.3 μmol/L for tetracaine. In non-pregnant myometrium, the IC50 was 26.9 μmol/L for bupivacaine, 40 μmol/L for ropivacaine, 384 μmol/L for lidocaine, and 7.4 μmol/L for tetracaine. These results suggested that local anesthetics do inhibit myometrial contractions in pregnant and non-pregnant rats in a concentration-dependent manner. Keywords: local anesthetics, bupivacaine, ropivacaine, lidocaine, tetracaine, pregnant, non-pregnant, myometrium, rat, in vitro
The goal of obstetric analgesia is to provide optimal pain relief for the parturient with minimal risk to herself and the fetus. Local anesthetic-induced neural blockade is the most effective means of reducing pain during labor and the postoperative period, and epidural analgesia is in widespread use for gynecologic and obstetric procedures. Compared with other forms of labor pain relief, epidural analgesia is the most effective but increases the risk of instrumental delivery (1, 16). Three of the local anesthetics, bupivacaine, ropivacaine and lidocaine are commonly used in obstetric analgesia, but tetracaine is used only as an adjunct in obstetric patients. Whether epidural analgesia with these anesthetics affects uterine contractility, directly or indirectly, is unclear. Determining the effects of local anesthetics on pregnant myometrium might be one way to assess their therapeutic value for preterm labor, while their effects on non-pregnant myometrium might address their therapeutic value for dysmenorrhea and gynecologic
Corresponding authors: Zi-Gang Li Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University Hangzhou, Zhejiang 310006, China Phone: 86-571-88208230; Fax: 86-571-88208231; E-mail: [email protected] 0231–424X/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
Depressant effects of local anesthetics on rat myometrium
229
surgery. However, to date, comparisons of the in vitro effects of local anesthetic agents on pregnant and non-pregnant myometrium are not well-documented. Previous in vitro studies have suggested that commonly-used local anesthetics alter the spontaneous contractility of pregnant uterine muscle, however, the results are inconsistent. Karsli et al. (9) suggested that bupivacaine at 300 μmol/L decreases the amplitude of contractions, but the frequency increases. Fanning et al. (5) reported similar results in human pregnant myometrium. On the contrary, Arici et al. (2) reported that bupivacaine had no effect on the frequency of uterine contractions, but inhibits rat myometrial contractility significantly more than ropivacaine at 10−4 mol/L. In this study, we evaluated the effects of four commonly-used local anesthetic agents, bupivacaine, ropivacaine, lidocaine and tetracaine (an ester anesthetic) on the contractions of myometrium isolated from pregnant and non-pregnant rats. Materials and Methods Animals Two-month-old, pregnant female Sprague-Dawley rats (n = 8) weighing 270–300 g at 18–20 days of gestation, and 2-month-old, non-pregnant female Sprague-Dawley rats (n = 10) weighing 200 ± 20 g were used. The latter were pretreated with 40 mg diethylstilbestrol subcutaneously for 2 days to induce estrus and improve the response to the drugs. The animals were cared for in accordance with the Guide for the Care and Use of Experimental Animals and the procedures were approved by the Ethics Committee of Zhejiang University School of Medicine. Experimental procedures Pregnant and non-pregnant rats were used for each local anesthetic. After anesthesia with urethane (25%, 4 ml/kg, intravenously), they were killed by cervical subluxation. The preparation of pregnant myometrial strips was performed according to the method described previously (12). The uterine horns were rapidly excised and carefully cleaned from the surrounding connective tissue and opened longitudinally along the mesenteric border. The fetuses of the late-stage pregnant rats were removed and non-uterine tissues were dissected away and discarded. Eight longitudinal full-thickness myometrial muscle strips (measuring 4 × 10 mm) were obtained from each pregnant rat. The uterus of the non-pregnant SpragueDawley rats was rapidly isolated, carefully cleaned of surrounding connective tissue, and 4 strips (10-mm long) were prepared. Eight pregnant and non-pregnant myometrial strips were studied for each local anesthetic. Each strip was mounted vertically, one end connected to the lower hook of the bath and the other to a force rod. The strips were incubated in a 10-ml tissue bath containing DeJalon’s solution (in mM/L: NaCl 153.9, KCl 5.6, glucose 2.7, NaHCO3 5.9, and CaCl2 0.27), which was aerated continuously with 95% O2 and 5% CO2. The pH was maintained at 7.4, and the temperature at 32 ± 0.5 °C to maintain the condition of the strips. The solution was made-up daily for each experiment. The pregnant myometrial strips were allowed to equilibrate at 1 g tension for 20 min until the contractions became regular, as previously reported (12). The characteristics of the contraction frequency and amplitude were recorded by a force displacement transducer (JZ100, Xinhang Machine and Equipment, Gaobeidian, China) coupled to a MedLab Biological Signal Collection System (Medease Science and Technology, Nanjing, China). When the contractions became regular, their frequency and amplitude were recorded as baseline activity, and the local anesthetics were added. Acta Physiologica Hungarica 101, 2014
230
Wei JS et al.
During epidural anesthesia or nerve block, the usual plasma concentrations of the anesthetics are < 100 μmol/L (3, 10, 18). Therefore, we used similar concentrations to compare the effects of the four local anesthetics on the contractility of pregnant and nonpregnant rat myometrium in vitro. Bupivacaine (Shanghai Harvest Pharmaceutical Co. Ltd, No. 4H04009), ropivacaine (Naropin, Astrazeneca AB, No. EF1026), lidocaine (Shanghai Fosun Zhaohui Pharmaceutical Co. Ltd, No. 050308), or tetracaine (Zhejiang Jiuxu Pharmaceutical Co. Ltd, No: 20050405) was added at cumulative concentrations from 0.01 μmol·L–1 to 300 μmol·L–1. Drug-containing solutions were prepared immediately before each experiment. The non-pregnant myometrial strips were allowed to equilibrate at 2 g tension for 40 min, after which contraction was stimulated by oxytocin (0.05 U/10 ml) as previously reported (12). When the contractions became regular, their frequency and amplitude were recorded as baseline activity. The drugs were then added and contraction frequency and amplitude were recorded as described above. The duration of exposure to each concentration was 15 min. The investigators were blinded as to the local anesthetic being used. Each strip was exposed to only one anesthetic agent. Control strips (without drug treatment) were recorded for the same time, and no significant decrement in contraction was observed. The primary end-point of the study was cessation of contractions. Statistical analysis All data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., USA). The concentration that caused 50% inhibition of contraction amplitude (IC50) was calculated by the LD50 data treatment sequence (Blue Cosmos Studio, China). P < 0.05 was considered statistically significant. A)
B)
Fig. 1. Effects of four local anesthetics on the amplitude (A) and frequency (B) of contractions of myometrial strips isolated from non-pregnant rats. Data (mean ± SEM) are expressed relative to baseline values; n = 8; *P < 0.05, **P < 0.01 vs. baseline; +: no contraction Acta Physiologica Hungarica 101, 2014
Depressant effects of local anesthetics on rat myometrium
231
A)
B)
Fig. 2. Effects of four local anesthetics on the amplitude (A) and frequency (B) of contractions of myometrial strips isolated from pregnant rats. Data (mean ± SEM) are expressed relative to baseline values; n = 8; *P < 0.05, **P < 0.01 vs. baseline; +: no contraction
Results In general, all the local anesthetics decreased the amplitude of contraction at the highest concentrations in both the pregnant and non-pregnant uterus (Figs 1A and 2A). The same was true for the frequency of contraction, except for the cases of bupivacaine and lidocaine in the pregnant uterus, which had no significant effect (Figs 1B and 2B). Bupivacaine effects From 1 to 30 μmol/L (pregnant strips), or 1 to 10 μmol/L (non-pregnant strips), bupivacaine did not significantly change the contractile amplitude. However, at 100 μmol/L, it depressed the amplitude in both non-pregnant and pregnant myometrium. In terms of frequency, there was a non-significant tendency to increase up to 30 μmol/L in pregnant myometrial strips (Fig. 2B), but the tendency was to decrease, with significant inhibition at 100 μmol/L, in the non-pregnant strips. Ropivacaine effects Ropivacaine significantly inhibited the amplitude of pregnant and non-pregnant myometrial contractions in a concentration-dependent manner. In pregnant strips, the range was from 1 to 100 μmol/L, and contractility almost ceased at 300 μmol/L. In non-pregnant strips, the range was from 10 to 100 μmol/L and contractions ceased completely at 300 μmol/L. However, ropivacaine tended to slightly increase the frequency at 100 μmol/L in pregnant strips, and at 0.1 μmol/L in non-pregnant strips, but the values did not significantly differ from baseline. Acta Physiologica Hungarica 101, 2014
Wei JS et al.
232
Lidocaine effects Exposure to lidocaine at 100–300 μmol/L significantly decreased the contraction amplitude in both strips. In pregnant strips, it had no effect on contraction frequency even at 300 μmol/L, but the frequency significantly decreased in the non-pregnant strips. Tetracaine effects Tetracaine caused a concentration-dependent inhibition of contractility in pregnant and nonpregnant uterine muscle. It depressed the amplitude of contractions from 30 to 100 μmol/L in pregnant strips, and from 1 to 100 μmol/L in non-pregnant strips (300 μmol/L abolished contractions). In pregnant strips, the response concentration was from 3 μmol/L, and contractions almost ceased at 100 μmol/L. In non-pregnant strips, the frequency was depressed only at 100 μmol/L. Comparison of local anesthetics Based on the above results, tetracaine had a significantly greater depressant effect than the other agents, whereas lidocaine had a significantly lower depressant effect than the others. In terms of IC50, the order of depressant potency in non-pregnant and pregnant myometrium was: tetracaine > bupivacaine > ropivacaine > lidocaine. Based on the IC50 values, the approximate relative local anesthetic potencies of equipotent doses of lidocaine, bupivacaine, ropivacaine and tetracaine were 60:6:4:1 (Table I). Table I. IC50 values for the four local anesthetics on contraction amplitude of pregnant and non-pregnant myometrial strips
IC50
Pregnant (μmol/L)
Non-pregnant (μmol/L)
Bupivacaine
100
26.9
Lidocaine
1771
384
Ropivacaine
157
40
Tetracaine
26.3
7.4
Discussion Bupivacaine is a long-acting local anesthetic of the amide type recommended for infiltration, peripheral nerve block, and epidural/spinal anesthesia. Ropivacaine is a long-acting, amidetype local anesthetic. Its structure and pharmacokinetics are similar to those of bupivacaine; however, ropivacaine has a better cardiotoxicity profile. Ropivacaine is also less lipid-soluble and clears via the liver more rapidly than bupivacaine. Due to its superior safety profile and significantly improved sensory-motor differentiation (11), ropivacaine is currently the longacting anesthetic of choice in our practice. Lidocaine, a short-acting, amino-amide local anesthetic, is not the most widely used in obstetric anesthesia in the USA, but it is the most widely used in China, due to its inherent potency, rapid onset, tissue penetration, effectiveness and cost-effectiveness. Tetracaine is an ester-type local anesthetic with an intermediate to long-duration mode of action. It is mainly used topically in ophthalmology and as an antipruritic, and it has been used in spinal anesthesia. All these local anesthetic agents have been used in obstetric anesthesia and analgesia. Acta Physiologica Hungarica 101, 2014
Depressant effects of local anesthetics on rat myometrium
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Previous studies suggested that bupivacaine causes a concentration-dependent decrease in the amplitude of contractions in pregnant and non-pregnant rat myometrium (2, 12, 14). Studies of human pregnant myometrium in vitro also gave similar results (5, 19). However, some in vivo clinical studies have suggested that bupivacaine has no effect on uterine contractility (15, 17). Furthermore, controversy exists regarding the issue of frequency. Arici et al. (2) reported that bupivacaine has no effect on the frequency of uterine contractions in pregnant 18- to 21-day-old Wistar rats. Karsli et al. (9) indicated that bupivacaine at 300 μmol/L increases the frequency of uterine contractions in albino rats pregnant for 18–21 days. Here, we found that bupivacaine inhibited the amplitude, and tended to increase the frequency in a concentration-dependent manner in pregnant myometrium, consistent with the Fanning et al. report (5), but at higher concentration (100 μmol/L) it had a slightly depressant effect. But in non-pregnant rat myometrium, bupivacaine decreased rather than increased the frequency, suggesting that non-pregnant myometrium may be more sensitive than pregnant myometrium. Our results suggested that ropivacaine had an effect similar to bupivacaine in pregnant and non-pregnant myometrium. Bupivacaine had a greater inhibitory effect on contractility than ropivacaine at higher concentrations (100 and 300 μmol/L), while ropivacaine had a greater inhibitory effect than bupivacaine at lower concentrations (1–30 μmol/L). Arici’s report also showed some similarities with our results (2). In terms of frequency, non-pregnant rat myometrium was more sensitive than pregnant myometrium at 300 μmol/L ropivacaine which completely inhibited the contraction of non-pregnant rat myometrium. A previous study suggested that lidocaine has a significantly greater depressant effect on spontaneous myometrial contractions in the human during late pregnancy than bupivacaine (19), but our results are in contrast to this finding. Based on the IC50, bupivacaine was 17-fold more potent than lidocaine. In pregnant rat myometrium, Fauza et al. demonstrated that lidocaine and ropivacaine both increased the contraction frequency at low concentrations along with a depression in amplitude (6, 7). In our study, lidocaine had no effect on the frequency. In non-pregnant rat myometrium, the results on amplitude were similar to those in pregnant rat myometrium, but on frequency, non-pregnant myometrium was more sensitive than pregnant myometrium at 300 μmol/L. Tetracaine has a recognized use during labor and delivery, but here are no reports recording the effects of the drug on duration of labor, incidence of forceps delivery, status of the newborn, and later growth and development of the child, as well as on the amplitude and frequency in pregnant or non-pregnant myometrium. Maruta and Osa (13) reported that tetracaine inhibited Ca2+ influx and K+ conductance in the uterine longitudinal muscle of estrogen-treated rats. Györke et al. (8) described tetracaine at low concentrations caused an initial inhibition of spontaneous Ca2+-release events in rat ventricular myocytes, while at high concentrations, it blocked release completely. In our study, tetracaine clearly decreased the amplitude and frequency in pregnant and non-pregnant myometrium. It had the highest potency among the four local anesthetics, and pregnant myometrium was more sensitive than non-pregnant myometrium in frequency, but non-pregnant myometrium was more sensitive than pregnant myometrium in amplitude. This suggested that tetracaine may be harmful in pregnancy. In the clinic, the concentrations of local anesthetics achieved in the human myometrium with normal administration in the epidural space are
