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Anesth Analg 2005;100:823-829 © 2005 International Anesthesia Research Society doi: 10.1213/01.ANE.0000145010.60144.ED
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Abstract |
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We investigated the effects of early and late sepsis on the actions of nondepolarizing neuromuscular blockers by using a rat sepsis model induced by cecal ligation and puncture. Isometric twitch tensions of nerve-hemidiaphragm preparations elicited by indirect (phrenic nerve) supramaximal stimulation at 0.1 Hz were evaluated. Rocuronium, pancuronium, and d-tubocurarine dose-dependently decreased the twitch tensions of the nonseptic, early septic, and late septic diaphragms (P < 0.01 each by analysis of variance [ANOVA]). Late sepsis shifted the concentration-twitch tension curves rightward from those of nonsepsis to larger degrees than did early sepsis, as indicated by increases in 50% inhibitory concentration (IC50) values (P < 0.01 each by ANOVA and P < 0.01 or 0.05 by the Scheffé F test). The standardized rightward shifts in early and late sepsis were largest for pancuronium, second largest for rocuronium, and smallest for d-tubocurarine (5.741, 2.979, and 1.660 times in late sepsis, respectively; P < 0.01 each by ANOVA and the Scheffé F test). Sepsis-induced increases in IC50 values did not accompany the decreases in slopes. The results indicate that sepsis induces hyposensitivities to nondepolarizing neuromuscular blockers, the degree of which depends on the stage of sepsis and on the kind of neuromuscular blocker.
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Introduction |
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Nondepolarizing neuromuscular blockers have frequently been used during management of anesthesia and intensive care for patients with sepsis. Sepsis has been reported to be one of the mechanisms associated with resistance to the actions of nondepolarizing neuromuscular blockers in burn injury1 (1,2).
It has been reported that the neuromuscular blocking action of d-tubocurarine is attenuated in vivo in animals with endotoxin-induced sepsis (3) and in vitro in muscles from animals with late sepsis (4–6) induced by surgical panperitonitis (7–10). However, the effects of sepsis on the actions of nondepolarizing neuromuscular blockers other than d-tubocurarine have not been determined. The stage dependency in the effects of sepsis on the actions of nondepolarizing neuromuscular blockers has also not been investigated.
The hypothesis of this study was that the sepsis-induced attenuations in the actions of nondepolarizing neuromuscular blockers depend on the stage of sepsis and on the kind of neuromuscular blocker. The aim of this study was to determine the differences in the effects of sepsis on the actions of nondepolarizing neuromuscular blockers and to determine the differences between the effects of early sepsis and late sepsis. We evaluated the neuromuscular blocking actions of rocuronium, pancuronium, and d-tubocurarine on twitch tensions of phrenic nerve-hemidiaphragm preparations removed from rats with early sepsis and late sepsis induced by panperitonitis and also from nonseptic rats as controls.
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Methods |
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The study was approved by the Animal Care and Use Committee of Sapporo Medical University. Adult male Wistar rats (7–9 wk old and weighing 255–295 g; n = 72) were randomly divided into the following 4 groups: 1) a group in which no procedure was performed (normal group; n = 18), 2) a group in which a cecal ligation and puncture (CLP) operation was performed and 9 h had passed since the operation (9h-CLP group; n = 18), 3) a group in which a CLP operation was performed and 18 h had passed since the operation (18h-CLP group; n = 18), and 4) a group in which a sham operation was performed and 18 h had passed since the operation (sham group; n = 18). In the 9h-CLP and 18h-CLP groups, sepsis was surgically induced by the CLP method (7); i.e., the cecum was ligated below the ileocecal valve and perforated in 3 locations with an 18-gauge needle via a midline abdominal incision under oxygen/isoflurane anesthesia. CLP is a representative animal septic model induced by bacterial panperitonitis from intestinal perforation, and its pathophysiology is similar to that of clinical intraabdominal sepsis (7,11). In a previous study using our CLP model, rats demonstrated septic changes in systemic organs, and their mortality rates were 25.0%, 33.3%, 50.0%, and 100% at 12, 18, 24, and 36 h after the operation, respectively (4). It has been reported that a "hyperdynamic and hypermetabolic state" and a "hypodynamic and hypometabolic state" are induced in a CLP animal within 10 and 12 h after the CLP operation, respectively (8–10). Therefore, we regarded CLP rats at 9 and 18 h after the operation to be in the stages of early sepsis and late sepsis, respectively (12). In the sham group, a similar midline abdominal incision was made under oxygen/isoflurane anesthesia, and the cecum was manipulated but not ligated or punctured. After the CLP or sham operation, each of the rats was subcutaneously injected with saline (5 mL/100 g of body weight) in the back for fluid resuscitation and was freely allowed water but no food.
The left hemidiaphragm with attached phrenic nerve, central tendon, and rib cage intact was rapidly removed from each of the rats that had been killed with excessively deep oxygen/isoflurane anesthesia and bled out. Strips of diaphragm (10 mm wide) with the phrenic nerve—which had been cut parallel to the muscle fibers—attached were dissected. Each isolated strip was mounted vertically in a tissue chamber (25 mL in volume), fixed at the rib cage, inferiorly positioned, and suspended at the central tendon from a force displacement transducer by using a 3-0 silk suture. The chamber was filled with modified Krebs solution maintained at 27°C and bubbled with 95% oxygen/5% CO2. The composition of the modified Krebs solution was (mM) NaCl, 118.0; KCl, 3.7; CaCl2, 2.5; MgCl2, 1.3; NaHCO3, 26.2; Na2HPO4, 1.2; and glucose, 11; pH was 7.40 ± 0.05 during bubbling.
Isometric twitch tension of the diaphragm was estimated by researchers who had not been informed of which experimental group each specimen belonged to. Twitch tension was elicited by indirect (phrenic nerve) or direct (muscle) supramaximal constant current stimulation at 0.1 Hz (for 0.05 and for 0.2 ms in the cases of indirect and direct stimulation, respectively) by using a stimulator and a constant-current unit with an optimal preload (2.8–5.5 g) to deliver maximal twitch tension. The phrenic nerve was positioned on wire bipolar platinum electrodes for indirect stimulation. For direct stimulation, a pair of plate bipolar platinum electrodes (each 25 x 12 mm square) was used, and the diaphragm was pretreated with a sufficient concentration (12 µM) of d-tubocurarine to completely abolish neuromuscular transmission. The twitch tension was recorded via the force transducer on a thermal chart recorder.
After the elicited twitch tension had been stabilized, single-twitch tension (averaged in groups of five) was determined. Then a drug was applied to the preparation extracellularly from the bathing solution. The drug concentrations were determined by adding a freshly prepared solution (dissolved in modified Krebs solution) of rocuronium (100 µM or 10 mM), pancuronium (10 µM or 1 mM), or d-tubocurarine (10 µM or 1 mM) with calibrated micropipettes into 25 mL of modified Krebs solution in the tissue chamber. After stabilization of the drug effect, single-twitch tension was again determined. Data were accepted only when twitch tension returned to 95%–105% of the initial value by rinsing the diaphragm preparation with modified Krebs solution in each study. Rocuronium bromide and pancuronium bromide were obtained from N.V. Organon (Oss, The Netherlands), and all other drugs were purchased from Sigma (St. Louis, MO).
Indirectly elicited twitch tension in the same preparation with no neuromuscular blocker was defined as the control value. Competition analysis data (50% inhibitory concentration [IC50] and slope at IC50) were determined from a four-variable logistic sigmoidal dose-response model fitted to the concentration-twitch tension curves (all values were considered for analysis) with the computer program Prism 4 (GraphPad Software, Inc., San Diego, CA). Data of twitch tension (in grams or percentage of the control value) are expressed as mean ± sd. Data of log IC50 (log µM), log IC50 ratio, and slope at log IC50 are expressed as mean ± sd. The IC50 ratio was defined as the IC50 of the concentration-twitch tension curve in the 9h-CLP or 18h-CLP group divided by that in the sham group. Standard errors of log IC50 ratios were calculated as [(log sesham)2 + (log seCLP)2]0.5, where log sesham and log seCLP are standard errors of log IC50 values of the sham and CLP groups, respectively. Data of IC50 values (in µM) and IC50 ratios are expressed as means with 95% confidence intervals. Statistical significance for IC50 values and IC50 ratios was calculated from log IC50 and log IC50 ratio, respectively. The two-tailed paired Student’s t-test, one- or two-way repeated-measures analysis of variance (ANOVA) with post hoc (Scheffé F) testing, and one-way factorial ANOVA with the Scheffé F test were used for statistical comparison, and P < 0.05 was accepted as significant.
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Results |
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The 9h-CLP and 18h-CLP rats, but not the normal and sham rats, demonstrated low levels of spontaneous activity, a decrease in escaping movements, and napped hair before dissection and also showed obvious pathologic changes in systemic organs (inflammatory ascites and edema of intestines, liver, kidney, lung, and so on), but not in diaphragms, on gross inspection on dissection.
The sizes of the diaphragm preparations used were mostly the same in all experimental groups (9–11 mm wide). The magnitudes of twitch tensions elicited by indirect and direct stimulations were largest in the sham group, second largest in the 9h-CLP group, and smallest in the 18h-CLP group (P < 0.01 each by ANOVA and the Scheffé F test; Fig. 1). There was no significant difference between twitch tension elicited by indirect or direct stimulation in the normal group and that in the sham group. There was no significant difference between the indirectly and directly elicited twitch tensions in each experimental group (Fig. 1).
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In each experimental group, there was no significant difference among the control twitch tensions elicited by indirect stimulations for rocuronium, pancuronium, and d-tubocurarine. After rinsing the diaphragms that had been exposed to one of these neuromuscular blockers with modified Krebs solutions, the indirectly elicited twitch tension returned to 95.4%–104.8% of the initial value in each study.
In the normal rats, rocuronium, pancuronium, and d-tubocurarine reduced the magnitudes of indirectly elicited twitch tensions dose-dependently (P < 0.01 each by ANOVA; Fig. 2). The concentration-twitch tension curves of these neuromuscular blockers were significantly different (P < 0.01 each by ANOVA and Scheffé F test; Fig. 2). IC50, which quantitatively indicates the position of the curve, was significantly largest for rocuronium, second largest for pancuronium, and smallest for d-tubocurarine (P < 0.01 each by ANOVA and Scheffé F test; Table 1). The slope for d-tubocurarine was significantly (P < 0.01 each by ANOVA and Scheffé test) smaller than those for rocuronium and pancuronium, which were not significantly different from each other (Table 1). For each neuromuscular blocker, there was no significant difference between the concentration-twitch tension curve, IC50, and slope in the normal group and those in the sham group (Figs. 2 and 3, Table 1).
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In the sham, 9h-CLP, and 18h-CLP groups, rocuronium, pancuronium, and d-tubocurarine reduced the magnitudes of indirectly elicited twitch tensions dose-dependently (P < 0.01 each by ANOVA; Fig. 3). For rocuronium and pancuronium, the concentration-twitch tension curves were significantly (P < 0.01 each by ANOVA) shifted from those of the sham group to the right in both the 9h-CLP group (P < 0.05 and 0.01, respectively, by Scheffé F test) and the 18h-CLP group (P < 0.01 each by Scheffé F test; Fig. 3, A and B). IC50 values for rocuronium and pancuronium were largest in the 18h-CLP group, second largest in the 9h-CLP group, and smallest in the sham group (P < 0.01 each by ANOVA and Scheffé F test; Table 1). The concentration-twitch tension curve for d-tubocurarine was significantly shifted from that of the sham group to the right in the 18h-CLP group (P < 0.01 each by ANOVA and Scheffé F test), but not in the 9h-CLP group (Fig. 3C). The IC50 for d-tubocurarine in the 18h-CLP group was significantly (P < 0.01 by ANOVA) larger than those in the sham and 9h-CLP groups (P < 0.01 and 0.05 by Scheffé F test, respectively; Table 1). For rocuronium, the slope in the 18h-CLP group was larger than those in the sham and 9h-CLP groups (P < 0.01 by ANOVA and P < 0.05 each by Scheffé F test), but there was no significant difference among the slopes for pancuronium and d-tubocurarine (Table 1).
The IC50 ratios, which standardize the magnitudes of the rightward shifts of the concentration-twitch tension curves, were significantly (P < 0.01 each by ANOVA) largest for pancuronium, second largest for rocuronium, and smallest for d-tubocurarine in both the 9h-CLP group (P < 0.01 or 0.05 each by Scheffé F test) and the 18h-CLP group (P < 0.01 each by Scheffé F test; Table 2).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results of this study primarily demonstrated that sepsis induced by panperitonitis stage-dependently and differentially attenuated the neuromuscular blocking actions of rocuronium, pancuronium, and d-tubocurarine on indirectly elicited twitch tension of the rat diaphragm in vitro.
The sepsis-induced attenuation of muscle contractility (13–16) advanced depending on the stage of sepsis, and the variation of attenuation was not large for each group, as indicated by the directly elicited twitch tensions and their standard deviations. Behavioral and pathologic findings seemed less varied in the early and late septic rats. These results suggest that the conditions of early sepsis in the 9h-CLP group and of late sepsis in the 18h-CLP group were similar when the rats were dissected.
In this study, the concentration-twitch tension curves of rocuronium, pancuronium, and d-tubocurarine shifted rightward in the septic rats but not in the sham and normal rats. These results clearly indicate that sepsis, but not surgical invasion, attenuates the actions of these neuromuscular blockers. Mechanisms associated with the sepsis-induced hyposensitivities to the neuromuscular blocking actions have not been investigated as extensively as mechanisms associated with muscle contractility, i.e., actions of septic inflammatory mediators, endotoxin, and septic oxygen-delivered free radicals (13–16). It is assumed that the endotoxin-induced increase in the probability of quantal acetylcholine release, which theoretically increases acetylcholine release from motor nerve terminals (17), may be a part of the mechanisms of the hyposensitivities to these neuromuscular blocking actions. Because the molecular structure of d-tubocurarine (a curare alkaloid) and those of pancuronium and rocuronium (bis-quaternary ammonium steroids) are obviously different, it is assumed that sepsis may also attenuate the actions of other nondepolarizing neuromuscular blockers that have not yet been revealed.
The rightward shifts of the concentration-twitch tension curves, as indicated by increases in IC50 values, in early sepsis were not as large as those in late sepsis for each of the neuromuscular blockers. Late sepsis-induced rightward shifts of the curves of nondepolarizing neuromuscular blockers other than d-tubocurarine (4) and early sepsis-induced rightward shifts of the curves were primarily demonstrated in this study. Because the condition of early sepsis in the 9h-CLP model is a transitory physiopathological state from nonsepsis to late sepsis, these results indicate that sepsis attenuates the neuromuscular blocking actions by degrees through the early phase to the late phase. In contrast to rocuronium and pancuronium, the rightward shift for d-tubocurarine in early sepsis was not significant. Because late sepsis significantly shifted the curve of d-tubocurarine rightward, it is thought that early sepsis may also have the potential to attenuate the action of d-tubocurarine. The small magnitude of sepsis-induced attenuation of the action of d-tubocurarine compared with the magnitudes of sepsis-induced attenuation of the actions of pancuronium and rocuronium might be the reason for the shift in the curve of d-tubocurarine of early sepsis being not significant.
Sepsis-induced increases in IC50 values did not accompany the decreases in the slopes; sepsis did not alter, or rather increased, the slopes. Nondepolarizing neuromuscular blocker-induced twitch depression at a low stimulus frequency reflects the competitive blocking of the postjunctional acetylcholine receptors. This strongly suggests that the sepsis-induced hyposensitivities to these neuromuscular blockers originated from an increase in the number of postjunctional acetylcholine receptors and not from a decrease in the affinities of the neuromuscular blockers to the receptors. Proliferation of acetylcholine receptors on the neuromuscular junction accompanying hyposensitivity to d-tubocurarine has been reported after burn injury in rats (18). Burn-induced hyposensitivities to nondepolarizing neuromuscular blockers require the condition of sepsis.1 These findings support the speculation based on the results of this study of an increase in the number of acetylcholine receptors. The increased slope in late sepsis for rocuronium suggests that sepsis may also have a concealed potential for increasing the affinities of the acetylcholine receptors to nondepolarizing neuromuscular blockers, at least to rocuronium, which theoretically and conversely increases sensitivity to the neuromuscular blockers. The abrupt onset of a steep downward slope of the curve in late sepsis for rocuronium indicates the possibility that sepsis facilitates the function of high-affinity binding sites of the acetylcholine receptors to nondepolarizing neuromuscular blockers (19).
The IC50 ratios in early and late sepsis were different among the neuromuscular blockers, thus indicating that the magnitude of sepsis-induced attenuation depends on the kind of neuromuscular blocker. The mechanisms underlying the differential attenuations have not yet been elucidated. The molecular structures of rocuronium and pancuronium are different from that of d-tubocurarine. Under the condition of nonsepsis, the slopes for rocuronium and pancuronium, which were very similar, were larger than the slope for d-tubocurarine. The IC50 ratios in early and late sepsis of rocuronium and pancuronium were larger than those of d-tubocurarine but were also different from each other. These results suggest that the sensitivities of the neuromuscular blockers to sepsis may, at least partially, depend on the molecular structures of the neuromuscular blockers.
Even though the results were obtained from animals, the findings seem to have major potential clinical relevance. Because the data were obtained in vitro, it is suspected that the sepsis-induced changes in the clinical actions of nondepolarizing neuromuscular blockers (1,2) may be associated with those in the pharmacodynamics in addition to those in the pharmacokinetics (2). The findings suggest that muscle weakness in clinical sepsis observed after recovery from nondepolarizing muscle relaxation is not caused by excessive sensitivity to the neuromuscular blocker but rather by impairment of skeletal muscle contractility (13–16). From the relevance in the conditions of early and late sepsis between the CLP model and clinical sepsis (8–11), it is assumed that clinical sepsis-induced hyposensitivity to nondepolarizing neuromuscular blockers may also advance by degrees through the stages of early sepsis to late sepsis. d-Tubocurarine may be less sensitive to clinical sepsis than recent nondepolarizing neuromuscular blockers of bis-quaternary ammonium steroids.
In summary, this study indicates that the magnitudes of sepsis-induced hyposensitivities to nondepolarizing neuromuscular blockers depended on the stages of sepsis and kinds of neuromuscular blockers. Late sepsis attenuated the actions of nondepolarizing neuromuscular blockers more intensely than did early sepsis. The sepsis-induced attenuations of the neuromuscular blocking actions were largest for pancuronium, second largest for rocuronium, and smallest for d-tubocurarine.
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Footnotes |
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1 Pavlin EG, Howard M, Slattory J. Sepsis increases resistance to nondepolarizing muscle relaxants in burned rats [abstract]. Anesthesiology 1996;85:A825.
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References |
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- Dwersteg JF, Pavlin EG, Heimbach DM. Patients with burns are resistant to atracurium. Anesthesiology 1986;65:517–20.[Web of Science][Medline]
- Thompson DF. Neuromuscular blocking agents in burn patients. DICP 1989;23:1006–8.[Medline]
- Tomera JF, Martyn J. Intraperitoneal endotoxin but not protein malnutrition shifts d-tubocurarine dose-response curves in mouse gastrocnemius muscle. J Pharmacol Exp Ther 1989;250:216–20.
[Abstract/Free Full Text] - Narimatsu E, Nakayama Y, Sumita S, et al. Sepsis attenuates the intensity of the neuromuscular blocking effect of d-tubocurarine and the antagonistic actions of neostigmine and edrophonium accompanying depression of muscle contractility of the diaphragm. Acta Anaesthesiol Scand 1999;43:196–201.[Web of Science][Medline]
- Nakayama Y, Narimatsu E, Sumita S, et al. Propofol enhances a d-tubocurarine-induced twitch depression in septic rat diaphragm. Anesth Analg 2000;90:80–4.
[Abstract/Free Full Text] - Narimatsu E, Aimono M, Nakayama Y, et al. The effects of midazolam and ketamine on D-tubocurarine-induced twitch depression in septic rat diaphragm. Res Commun Mol Pathol Pharmacol 2000;108:369–80.[Medline]
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[Abstract/Free Full Text] - Wilcox P, Milliken C, Bressler B. High-dose tumor necrosis factor alpha produces an impairment of hamster diaphragm contractility. Am J Respir Crit Care Med 1996;153:1611–5.[Abstract]
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[Abstract/Free Full Text] - Arias HR. Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors. Neurochem Int 2000;36:595–645.[Web of Science][Medline]
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