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Resistance to rocuronium of rat diaphragm ascompared with limb muscles

Lina Huang, MD, Meirong Yang, MD, Lianhua Chen, MD, PhD,and Shitong Li, MD, PhD*

Department of Anesthesiology, The Affiliated First People’s Hospital, Shanghai Jiaotong University, Shanghai, China

a r t i c l e i n f o

Article history:

Received 13 February 2014

Received in revised form

3 June 2014

Accepted 9 July 2014

Available online 16 July 2014

Keywords:

Potency

Rocuronium

Diaphragm

Extensor digitorum longus

Soleus

* Corresponding author. Department of Ane200080, China. Tel.: þ86 21 63240090 3023; fa

E-mail address: lishitongpaper@gmail.com0022-4804/$ e see front matter ª 2014 Elsevhttp://dx.doi.org/10.1016/j.jss.2014.07.017

a b s t r a c t

Background: Skeletal muscles are composed of different muscle fiber types. We investigated

the different potency to rocuronium among diaphragm (DIA), extensor digitorum longus

(EDL), and soleus (SOL) in vitro as well as to investigate the differences of acetylcholine

receptors (AChRs) among these three typical kinds of muscles.

Materials and methods: The isolated left hemidiaphragm nerve-muscle preparations, the EDL

sciatic nerve-muscle preparations, and the SOL sciatic nerve-muscle preparations were

established to evaluate the potency to rocuronium. Concentration-response curves were

constructed and the values of IC50 were obtained. The density of AChRs at the end plate

and the number of AChRs per unit fiber cross fiber area (CSA), AChR affinity for muscle

relaxants were evaluated.

Results: The concentration-twitch tension curves of rocuronium were significantly

different. The curves demonstrated a shift to the right of the DIA compared with the EDL

and SOL (P < 0.01), whereas no significant difference was observed between EDL and SOL

(P > 0.05). IC50 was significantly largest in DIA, second largest in SOL, and smallest in EDL

(P < 0.05). The number of AChRs per unit fiber CSA was largest in DIA, second largest in

EDL, and smallest in SOL (P < 0.01 or P < 0.05). The DIA showed the lowest affinity of the

AChRs, whereas the SOL showed the highest affinity.

Conclusions: The resistance to rocuronium of DIA compared with EDL and SOL was verified.

The DIA was characterized by the largest number of AChRs per unit fiber CSA and the

lowest affinity of the AChRs. Although compared with SOL, EDL was proved to have larger

number of AChRs per unit fiber CSA and the lower affinity of the AChRs. These findings may

be the mechanisms of different potency to rocuronium in DIA, EDL, and SOL. The results of

the study could help to explain the relationship between different composition of muscle

fibers and the potency tomuscle relaxants. Extra caution should be taken in clinical practice

when monitoring muscle relaxation in anesthetic management using different muscles.

ª 2014 Elsevier Inc. All rights reserved.

1. Introduction and type II x). Previous studies have reported that neuro-

Skeletalmuscles are composed of differentmuscle fiber types,

which includes type I and type II (comprising type II a, type II b,

sthesiology, The Affiliatedx: þ86 21 63069481.(S. Li).

ier Inc. All rights reserved

muscular junction (NMJ) morphology varies across muscle

fiber types. For example, NMJs at type I and IIa fibers are

smaller and less complex compared with NMJs at type IIx

First People’s Hospital, Shanghai Jiaotong University, Shanghai

.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 4 7 1e4 7 9472

and/or IIb fibers [1]. NMJ is the junction between a motor

neuron and a muscle fiber, which directly modulates muscle

activity by conducting nerve impulse to induce muscle con-

tractions [2]. NMJ achieving its physiologic functions depends

on its presynaptic and postsynaptic factors. Presynaptic fac-

tors include the modulation of acetylcholine (ACh) release

from motor nerve terminals, whereas postsynaptic factors

include acetylcholine receptors (AChRs) and the rate of ACh

hydrolysis by acetylcholinesterase (AChE) [3]. AChRs accept

ACh released from the nerves, thus evoking amuscle impulse.

Findings of previous clinical practices have indicated that

different monitoring positions for muscle relaxation in anes-

thetic management represented different extent of neuro-

muscular blockade. Some clinical reports showed that the

orbicularis oculi muscle was more resistant to non‑depola-

rizing muscle relaxants (NDMR) than the adductor pollicis

muscle [4]. In addition, Saitoh et al. [5,6] found that neuro-

muscular block recovered more quickly in the great toe than

in the thumb after the administration of vecuronium in their

series of reports. They also revealed that the mechanism un-

derpinning this observation was that the flexor hallucis brevis

muscle contains more type II fibers than the adductor pollicis

muscle [7] and type Imuscle fiber wasmore resistant to NDMR

[5,8e10]. Additionally, compared with the adductor pollicis

muscle, dose-response curves of the diaphragmatic response

demonstrated a shift to the right [11,12]. Furthermore, the

recovery of the diaphragm (DIA) -evoked response occurred

earlier than at the adductor pollicis muscle [13,14]. Besides the

factors thatmight influence themetabolism and absorption of

NDMR such as blood flow, oxygen consumption, and distance

from the heart and heart output [15,16], different muscle fiber

composition of these muscles was a well-known explanation

to these findings. However, there are contradictory opinions

about the relationship between the neuromuscular blockade

and the composition of muscle fibers in different muscles. As

we have mentioned above, type I muscle fiber is more resis-

tant to NDMR [17,18]. Somuscleswith higher contents of type I

muscle fibers are more resistant to NDMR than those with

higher type II muscle fibers [19]. Chen et al. [3,20] reported that

chronic dexamethasone treatment led to desensitization of

the rat DIA to rocuronium and that susceptibility to rocuro-

nium associated with decreased muscle fiber type II. Never-

theless, there are other reports demonstrated that muscle

type composition cannot explain the difference in muscle

relaxation effect [3,21]. Some researchers drew a conclusion

that there was no association between the composition of

muscle fiber types and sensitivity to muscle relaxants [21,22].

Zhou et al. [21] showed that the orbicularis oris muscle con-

tained fewer type I fibers than the gastrocnemius muscle but

displayed more desensitization to rocuronium. The density of

AChRs at the end plate and the number of AChRs per unit fiber

cross-section area (CSA), AChRs affinity for muscle relaxant

may play the most important role in the sensitivity of skeletal

muscles to NDMR [21].

Therefore, the aim of the present study was to investigate

the different responses to rocuronium among DIA (represents

mixed muscles), extensor digitorum longus (EDL) (with pre-

dominantly fast-twitch fibers), and soleus (SOL) (with pre-

dominantly slow-twitch fibers) in vitro as well as to investigate

the differences of AChRs among these three typical kinds of

muscles. We compared the density of AChRs at the end plate

and the number of AChRs per unit fiber CSA, AChRs affinity for

muscle relaxants. Therefore, the purpose of the present study

was to draw inferences about the relationship between the

functional results and morphologic changes.

2. Materials and methods

2.1. Animals

The study was approved by the Animal Care Committee in

Shanghai Jiaotong University (Shanghai, China). Twenty male

SpragueeDawley rats (Experimental Animal Center of the

School of Medicine, Shanghai Jiaotong University, Shanghai,

China), weighing 200e240 g, were housed in groups of three.

They were fasted but allowed to have free access to water and

food before the experiments.

2.2. Muscle preparations

12 rats were killed with 60 mg/kg pentobarbital intraperito-

neally. The isolated left hemidiaphragm nerve-muscle prep-

arations, the EDL sciatic nerve-muscle preparations, and the

SOL sciatic nerve-muscle preparations were established for

indirectly electrical stimulation as described previously

[23e25]. Body temperature was maintained at 37�C using a

heating blanket and radiant heat.

The left hemidiaphragm with attached phrenic nerve,

central tendon, and rib cage intact was rapidly removed from

each of the rats to investigate rocuronium potency. The right

hemidiaphragm were rapidly removed for morphologic anal-

ysis. Either the EDL or the SOL was exposed in one leg. After

measurements were completed for one muscle, the other was

then exposed in the other leg. The SOL was exposed by

sectioning the tendons connecting the plantaris and gastroc-

nemius muscles to the heel and reflecting the muscles back.

Silk thread was attached to the distal tendon of the SOL and

the tendon was sectioned. The muscle was then carefully

freed of surrounding tissues, ensuring the blood supply

remained intact, and the sciatic nerve was sectioned. The EDL

was prepared in the similar manner after first exposing the

muscle by reflection of the anterior tibialis muscle. The silk

sutures were tied to the proximal and distal tendons of the

EDL and/or SOL muscles and the muscles were removed,

tendon to tendon. The EDL or the SOLmuscles on the other leg

were removed simultaneously for morphologic analysis.

The isolated nerve-muscle preparations were dipped

immediately into Plexiglass chambers (ALC-M System for

Isolated Tissue-Organ Research; Shanghai Alcott Biotech,

Shanghai, China; 40 mL in volume) filled with Krebs solution,

maintained at 37�C, and bubbled with 95% oxygen and/or 5%

CO2. The composition of the Krebs solution was as follows:

137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM

KH2PO3, 12 mM NaHCO3, and 6.5 mM glucose, with a pH

7.40 � 0.05 during bubbling.

EDL, SOL, andDIA strips of the other sidewere dissected for

ATPase staining. The strips for ATPase staining were

embedded separately in plastic holders and rapidly frozen in

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 4 7 1e4 7 9 473

isopentane cooled in liquid nitrogen (�160�C), followed by

further freezing in liquid nitrogen.

2.3. Rocuronium potency in isolated muscular strips

The indirect electrical stimulation-evoked twitch tension was

recorded with MPAMultiple Channel Biological Signal Analysis

System (provided by the Department of Anesthesiology,

Shanghai First People’s Hospital of SJTU). Each isolated strip

was mounted vertically in a tissue chamber, inferiorly posi-

tioned. One side of the isolated hemidiaphragm preparation

was fixed at the rib cage, meanwhile, the other side was sus-

pended at the central tendon from a force displacement

transducer using a 3-0 silk suture (ALC-M System for Isolated

Tissue-Organ Research; Shanghai Alcott Biotech, Shanghai,

China; 40 mL in volume). As for the EDL and SOL preparations,

they were aligned vertically with distal tendon attached to the

force displacement transducer, proximal tendon fixed to the

stainless steel fixed-post. The chamber was filled with Krebs

solution as mentioned previously. The nerves of the prepara-

tions were positioned on wire bipolar platinum electrodes for

indirect stimulation. Isometric tension was elicited by indirect

(phrenic nerve or sciatic nerve) supramaximal constant voltage

stimulation at 0.1 Hz for 0.05 ms, using a stimulator and a

constant voltage unit. The twitch tension was recorded via the

force transducer on a recorder (ALC-MPA 2000m, Acquisition

andAnalysis System for Life Science Research, Shanghai Alcott

Biotech, Shanghai, China). The stimulator was activated by a

personal computer. Twitch stimuli were used to determine the

optimal length (L0) at which skeletal muscle may generate the

greatest force, followed by a 15-min thermo-equilibration

period. A number of measurements were performed as

described in subsequent sections.

After the elicited twitch tension had been stabilized for a

minimum of 15 min, the single-twitch tension, averaged in

groups of five, was determined. The proximal end of the nerve

attached to the strips was stimulated with a single supra-

maximal train of rectangular pulses (stimulation intensity,

which is the minimus stimulation intensity that can induce

maximal force of contraction, duration 0.2 ms). The pulse was

repeated three times at 5-s intervals, and the mean muscular

tension amplitude was calculated. Before rocuronium was

added in, the baseline twitch tension amplitudewas recorded.

Rocuronium was then applied to the preparation at the con-

centration of 0.01, 0.1, 0.5, 1, 2, 5, 7, 10, 12, 15, 20, 30, 40, and

50 mmol/L. Drug concentrations were determined by adding

freshly prepared solutions with calibrated micropipettes to

modified Krebs solution (40 mL) in the tissue chamber. After

stabilization of the drug effect for a minimum of 10 min,

single-twitch tension was again determined. Data were

accepted when twitch tension returned to 95%e105% of the

initial value by rinsing the DIA preparation with Krebs solu-

tion in each study. Rocuronium bromide was obtained from

N.V. Organon (Oss, The Netherlands) and all other drugs were

purchased from SigmaeAldrich (St. Louis, MO). Indirectly eli-

cited twitch tension in the same preparation with no neuro-

muscular blocker was defined as the control value.

Concentration-response curves were constructed and the

values of IC50 were obtained.

2.4. AChR evaluation

A total of 8 rats were killed as mentioned previously. Excised

rat DIA, EDL, and SOL muscles were pinned in the silicone-

lined organ bath with the aid of mini pins. A careful

dissection was performed under the microscope to isolate

small muscle strips along the length of the muscles. Thin

strips of the muscles were fixed for 1 h in 4% para-

formaldehyde. After fixation, each muscle strip was further

equally separated into smaller strands (each containing

5e10 single intact fibers) and then stained by AChE to

identify the motor end plates. Using a camera lucida mi-

croscope (Leica DMIRB; Leica Camera AG, Solms, Germany),

end plate areas of dissociated fibers were photographed

under the dissecting microscope (�100 magnification). The

mean end plate surface area (ESA) value (square microme-

ters) of muscles in each rat was obtained from the average

of the values in the dissociated strands (each containing 10

single fibers) [21].

A radioimmunoassay method using 125I-labeled AChR

competitive inhibitor a-bungarotoxin (125I-aBTX) followed

with gamma spectroscopy was used to detect the number of

AChRs at the end plates of each kind of muscle fiber. Another

five dissociated strands of differentmuscles were treatedwith

different concentrations of rocuronium (0, 2.5, 5.0, 7.5, and

10.0 mM). Then the strands were incubated in lactated Ringer

solution containing 125I-aBTX at the concentration of 1.0 mM

for 60 min at the room temperature. After washing several

times, the strands were fixed 1 h in 4% paraformaldehyde,

then the AChE staining as mentioned previously were pro-

ceeded. The intensity of radiation at the end plates was

measured. The difference in radioactivity between junctional

and extrajunctional segments was used to calculate the

number of 125I-aBTX binding sites per end plate. The number

of AChR per end plate in the absence of rocuronium was

defined as AChR0. The number of the free AChRs per end plate

under a series of experimental concentrations of rocuronium

was defined as AChRE. The mean density of AChRs on end

plates was obtained by dividing AChR0 by mean ESA (AChR0/

ESA). The degree of saturation of AChRs by the muscle

relaxant under the concentrations of rocuronium was calcu-

lated by (AChR0�AChRE)/AChR0 and presented as a percent-

age; this number reflects the affinity of AChR for the muscle

relaxant at each NMJ.

2.5. Muscle histologic studies

Muscle blocks were serially sliced at 15 mmon a cryostat (Leica

CM1850, Nussloch, Germany). The modified ATPase staining

method was applied in addition to hematoxylin and eosin

staining.

Muscle fiber types were classified as type I and II based on

their staining for myofibrillar ATPase following alkaline pH

10.4 and acid pH 4.5 using a modification of the procedure

described by Brooke and Kaiser [26]. Fibers were classified into

type I (slow-twitch muscle fiber with weak mATPase activity)

or type II (fast-twitch muscle fiber with strong mATPase ac-

tivity) [27]. The image analysis software Image-Pro Plus,

version 5.1 (Media Cybernetics, Silver Spring, MD) was used to

evaluate the prepared samples. Then, approximately 400

Fig. 1 e Twitch tension height in DIA, EDL, and SOL.

Statistical analysis was performed by two-way ANOVA

with post hoc Bonferroni testing. *P < 0.05 versus DIA.&P < 0.05 versus EDL.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 4 7 1e4 7 9474

fibers per muscle were measured for muscle fiber type distri-

bution and CSA (square micrometers) of each fiber type. The

distribution of muscle fibers of a given fiber type (type I or II)

was expressed as a percentage of all the fibers in the section.

2.6. Statistical analysis

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

Fig. 2 e Concentration-twitch tension curves for rocuronium (A

Curves of concentration-twitch tension in DIA, EDL, and SOL. Va

analysis was performed by two-way ANOVA with post hoc Bon

rightward in DIA compared with EDL and SOL (P < 0.01), where

and SOL (P > 0.05).

considered for analysis) with the computer program Prism 4

(GraphPad Software, Inc, San Diego, CA).

All other data were analyzed by SPSS version 13.0 software

(IBM, Armonk, NY). Data are expressed as mean � standard

deviation. One-way analysis of variance (ANOVA) was used to

test the significance of differences among all groups. The

paired t-test was used for comparison between any two

groups. P < 0.05 was considered to indicate a statistically

significant difference.

3. Results

3.1. Rocuronium potency in isolated muscular strips

Twelve standard deviation rats, weighing 200e240 g, were

killed and the isolated nerve-muscle strips were established

for indirectly electrical stimulation. The magnitudes of twitch

tensions elicited by indirect stimulations were largest in the

DIA group, second largest in the EDL group, and smallest in

the SOL group (P < 0.05 each by ANOVA; Fig. 1). After rinsing

the muscle strips that had been exposed to rocuronium with

modified Krebs solutions, the indirectly elicited twitch tension

returned to 95%e105% of the initial value in each study. In all

the groups, rocuronium reduced the magnitudes of indirectly

elicited twitch tensions dose dependently (P < 0.01 each by

ANOVA; Fig. 2). The concentration-twitch tension curves of

rocuronium were significantly different. The curves were

significantly shifted rightward in the DIA compared with the

EDL and SOL (P < 0.01 each by ANOVA; Fig. 2), whereas no

significant difference was observed between the EDL and SOL

(P > 0.05 each by ANOVA; Fig. 2). IC50, which quantitatively

) DIA, (B) EDL, and (C) SOL (*P < 0.01 versus control). (D)

lues are expressed as mean ± standard deviation. Statistical

ferroni testing. (D)The curves were significantly shifted

as no significant difference was observed between the EDL

Fig. 3 e ATPase staining of DIA (A), EDL (B), and SOL (C) at pH 4.6. The light-colored fibers are type 2 fibers, whereas the deep-

colored fibers are type 1 fibers (3100 magnification).

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 4 7 1e4 7 9 475

indicates the position of the curve, was significantly largest in

DIA, second largest in SOL, and smallest in EDL (P < 0.05 each

by ANOVA; Table 1). The slope for rocuronium in SOL was

significantly smaller than that in DIA and EDL (P< 0.05 each by

ANOVA), which were not significantly different from each

other (Table 1).

3.2. End plate AChR evaluation

One end plate was found in each fiber through AChE staining

for AChRs under the dissecting microscope (�100 magnifica-

tion); the end plates appeared to be arrayed like a string of

beads (Fig. 4). The ESA manifested significant differences

among the three kinds of muscles. ESA were largest in the

EDL, second largest in the DIA, and smallest in the SOL

(P < 0.05 each by ANOVA; Table 3).The mean density of AChR

at the end plate were significantly different in three muscles.

The density of AChR presented a decreased trend from the

DIA to the SOL and bottomed in the EDL (P < 0.05 each by

ANOVA; Table 3).

The degree of saturation of AChRs increased in a dose-

dependent manner in all three kinds of muscles as the con-

centration of rocuronium increased.Within each concentration,

the increase in theDIAwas significantly smaller than that in the

EDL and SOL (P < 0.05 each by ANOVA; Table 4).

3.3. Morphologic examination of muscle fibers

Morphologic composition was observed to be of significant

differences among the threemuscles. The percentage of type I

Table 1 e IC50 values and slopes of the concentration-twitch te

Rocuronium DIA

Log IC50 1.003 � 0.009

IC50 10.03 (9.590e10.43)

Slope �3.946 � 0.278

Log IC50, IC50, and slope at log IC50 were determined from a four-variable

twitch tension curves. The twitch tension was elicited by indirect stimul

intervals in IC50 (micrometer) andmean � standard deviation in log IC50 (lo

significance in IC50 was calculated from log IC50. Statistical analysis was*P < 0.05 versus DIA.y P < 0.05 versus EDL.

fibers was largest in the SOL, second largest in the DIA, and

smallest in the EDL but with the percentage of type II fibers

opposite of it (P< 0.05 each by ANOVA; Table 2; Fig. 3). The CSA

of type I fibers showed a significant incremental trend from

the DIA group to the EDL group and peaked in the SOL group

(P < 0.05 each by ANOVA; Table 2; Fig. 3). The CSA of type II

fibers also presented the same trend with significant differ-

ences between SOL group and the other two groups (P < 0.05

each by ANOVA; Table 2; Fig. 3). Mean fiber CSA manifested

significant differences among the three groups of muscles.

The DIA muscle was composed of fibers with a smallest CSA,

whereas the SOLmuscle was composed of fiberswith a largest

CSA, and the EDL muscle with middle CSA (P < 0.05 each by

ANOVA; Table 3; Fig. 3). Combined with morphologic analysis,

the number of AChRs per unit fiber CSAwas largest in the DIA,

second largest in the EDL, and smallest in the SOL (P < 0.01 or

P < 0.05 each by ANOVA; Table 3).

4. Disccussion

In our present study, the experiments were performed in the

rat SOL (slow-twitch), EDL (fast-twitch), and DIA (mixed

twitch). The DIA, one kind of respiratory muscles, is an

important skeletal muscle, which plays a pivotal role in

breathing. Because of the easiness to establish the prepara-

tions and its vital physiological actions, the DIA was widely

studied as a special object in many investigations on skeletal

muscles. As for EDL and SOL muscles, the somatic limb

nsion curves of rocuronium.

EDL SOL

0.824 � 0.017 0.857 � 0.024

7.190 (6.428e8.042)* 6.669 (6.167e7.213)*

�3.744 � 0.4764 �2.987 � 0.3871*,y

logistic sigmoidal dose-response model fitted to the concentration-

ation at 0.1 Hz. Values are expressed as means with 95% confidence

garithm ofmicrometer), and slope at log IC50 (n¼ 10 each). Statistical

performed by one-way ANOVA with post hoc Bonferroni testing.

Table 2 e Muscle fiber distribution and CSA.

Muscles Fiber-type proportion (% total) CSA (mm2)

Type I Type II Type I Type II

DIA 44.4 � 6.1 55.6 � 3.9 1959.6 � 495.6 3000.5 � 1271.9

EDL 5.5 � 0.56* 94.5 � 4.3* 2950.0 � 430.2* 3289.2 � 1224.7

SOL 92.2 � 8.1*,y 7.8 � 0.8*,y 4933.3 � 1865.8*,y 3673.8 � 483.4*,y

CSA ¼ cross section area.

Values are expressed as mean � standard deviation. Statistical analysis was performed by two-way ANOVA with post hoc Bonferroni testing.*P < 0.05 versus DIA group.yP < 0.05 versus EDL group.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 4 7 1e4 7 9476

muscles, representing typical fast and slow muscles, were

usually investigated as well.

The magnitudes of twitch tensions elicited by indirect

stimulations on nerves were largest in the DIA, second largest

in the EDL, and smallest in the SOL. Muscle activity is directly

modulated by nerve impulses from motor neurons via axons

to muscle fibers to induce muscle contractions. NMJs include

AChRs, which accept ACh released from the nerves, thus

evoking a muscle impulse. Different nerves release ACh

differently under the same stimulation [28]. Differences in

quantal transmitter release have been reported between

nerve terminals innervating rat fast and slow muscles [29].

The authors observed that the normal quantal content

determined in isolated nerve-muscle preparations of rat SOL

and EDL muscles was higher in EDL than that in SOL. The EDL

releasedmore than twice asmuch transmitter per unit area of

synaptic contact than the SOL. In the absence of any differ-

ence in the postsynaptic organization, it appeared that the

increased transmitter release accounted almost entirely for

the greater safety factor in the EDL. Thus quantal transmitter

release of ACh may contribute partly to the contractile force.

The quantal release of ACh is Ca2þ dependent. Ca2þ influx

trigger the action [30]. Nguyen-Huu et al. [3] reported the in-

crease in quantal release in the DIA with respect to the EDL.

And this may be due either to differences in the available

number of nerve terminal release sites and/or nerve terminal

extension [31] or to differences in calcium channels available

for triggering evoked quantal ACh release [32]. In the rat DIA,

extracellular Ca2þ and Ca2þ from the SR is required for

contraction in contrast to other skeletal muscles where only

SR Ca2þ is important [33]. The difference in quantal release of

ACh can explain the difference in magnitudes of twitch ten-

sions in three muscles.

Fig. 4 e AChE staining of end plate area of DIA (A), EDL (B), and S

muscle fiber display light-colored (3100 magnification).

The IC50 of rocuronium among DIA, EDL, and SOL were

taken as measures of muscle sensitivity. Our results IC50 of

DIA to rocuronium (10.03 mM), which demonstrated most

resistance to NDMR, was consistent with the largest number

of AChR per unit fiber CSA (2.8 � 103 mM�2) and lowest affinity

to rocuronium. Thus sensitivity was highest in the SOL (IC50

6.669 mM), which had the lowest number of AChR per unit fiber

CSA and higher affinity to rocuronium. Moreover, the shift of

concentration-twitch tension curves of DIA, EDL, and SOL to

the right indicated the increases of IC50 values. The increase in

IC50 accompanying the increase in the slopes, indicated the

lower affinities of the AChRs, which represented theoretically

hyposensitivity to NDMR. Above all, the findings were

consistent in our experiment.

Our results comparing the potency to NDMR between the

DIA and peripheral muscles were supported by the results of

previous studies [11,12]. These investigators compared the

effective NDMR in human subjects for the DIA and adductor

pollicis muscle by a cumulative bolus-dose method. They

found that the potency of NDMR was lower in the DIA than in

peripheral muscles [11,12]. Also, in previous experiments

NDMR in animal models were studied, either in vivo or in vitro.

The studies indicated that the DIA was more resistant to the

NDMR, as compared with peripheral muscles [3,34]. However,

the values of IC50 calculated in the present study were not

completely accordant with previous findings. Itoh et al. [35]

estimated neuromuscular effects of rocuronium by direct

measurement of twitch tension with a force transducer using

a cumulative infusion dosing method where a 2.5- to 3-fold

potency ratio of as DIA to the tibialis anterior muscle was

observed in vivo in rat. While in Nguyen-Huu et al. [3] study,

they compared the IC50 in DIA and EDL to d-tubocurarine, and

7-fold effective concentration ratio was observed. The twitch

OL (C).The end plates display brown and circle, whereas the

Table 3 e Morphologic data of DIA, EDL, and SOL.

Muscles ESA mm2 Density of AchRs on end plates �103 Mean fiber CSA, mm2 AchRs per unit fiber CSA �103

DIA 1186.0 � 329.9 7.2 � 2.9 2610.6 � 879.8 2.8 � 1.0

EDL 1966.8 � 867.2* 3.4 � 1.2* 3270.4 � 1023.1* 1.0 � 0.4*

SOL 884.4 � 444.5*,y 4.0 � 1.6*,y 4834.7 � 1216.7*,y 0.8 � 0.3*,y

CSA ¼ cross section area.

Values are expressed as mean � standard deviation. Statistical analysis was performed by two-way ANOVA with post hoc Bonferroni testing.*P < 0.01 versus DIA.y P < 0.05 versus EDL.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 4 7 1e4 7 9 477

tension measurements were performed in rat strips of hemi-

diaphragm and EDL nerve-muscle preparation in vitro. How-

ever, the study of Ibebunjo et al. [36] showed that the ED50 of

vecuronium in DIA was higher than SOL (13.8 versus 10.3 mg/

kg), which was performed in cats in vivo using the repeated

bolus dose-response method. The differences may be due to

the different methodology, various equipments, different

NDMR, and varied species studied which cannot be neglected

comparing the varied results. To the best of our knowledge, no

studies have been reported comparing the potency of

rocuronium among rat DIA, EDL, and SOL simultaneously

under the same circumstance.

Themuscleswe studied in the present study demonstrated

quite different compositions of muscle fiber types. The per-

centages of type I fiber in the EDL, SOL, and DIA muscles were

5.49 � 0.56%, 92.21 � 8.1%, and 44.40 � 6.1%, respectively

(Fig. 1), and showed a reduced distribution of type II fibers

from the EDL, the DIA to the SOL, indicating a shift toward

slow fibers. The observation is consistent with previous

studies. Apparently if the hypothesis thatmuscleswith higher

contents of type I muscle fibers were more resistant to

neuromuscular-blocking agents than those with higher type II

muscle fibers was true, the morphologic results cannot

explain our results about the potency of rocuronium in three

muscles, as IC50 was significantly largest in DIA, second

largest in SOL, and smallest in EDL.

Considering the different sensitivities to non-depolarizing

muscle relaxants between different muscles, the mechanisms

have been proved various. Perfusion, temperature, size of the

end plate, size of muscle fiber, and fiber composition may un-

derlie the differences in responses to neuromuscular-blocking

drugs. In the present study, we used isolated nerve-muscle

Table 4 e Degree of saturation under differentconcentrations of rocuronium in DIA, EDL, and SOL.

Muscles Rocuronium concentrations (mM)

0 2.5 5 7.5 10

DIA 0 23 � 10 32 � 9 40 � 8 51 � 8

EDL 0 28 � 9* 45 � 8* 48 � 8* 62 � 11*

SOL 0 30 � 10* 47 � 6* 54 � 11*,y 63 � 8*

Values are expressed as mean � standard deviation. Statistical

analysis was performed by two-way ANOVA with post hoc Bonfer-

roni testing.*P < 0.01 versus DIA.y P < 0.05 versus EDL.

preparations in organ bath under the stable temperature.

Therefore, the factors such as perfusion and temperature

should be excluded. Previous studies suggested that NMJs in

mammalian fast-twitch muscle fibers have more extensive

folds than in slow-twitch fibers [37]. Nevertheless, Wood and

Slater’s [29] morphometric analysis of NMJs in the EDL and SOL

muscles showed that the extent and spacing of folds playing

a vital part in ensuring reliable transmission during normal

activity was very similar in the two muscles. The reduction

of folding in itself would lead to a significant decrease in

the efficacy of neuromuscular transmission. Apparently, the

controversial findings cannot explain different sensitivities to

NDMR.

The relationship between neuromuscular responses and

size of muscle fiber was noted that the time to spontaneous

25% recovery but not potency to both depolarizing and non-

depolarizing drugs increased with fiber size [22]. Also, the

duration of blockade was proved to be shorter in muscles

comprised of small fibers with large end plates relative to fiber

size [38].

The size of the end plate and the number or density of

AChRs was insufficient to explain different potency to NDMR

among the muscles. The number of AChRs per unit fiber CSA

may be one of themorphologic factors causing the differential

responses to neuromuscular-blocking drugs between DIA,

EDL, and SOL, according to the result that sensitivity to

rocuronium was greater in DIA, which was comprised of

larger fibers with smaller number of AChRs relative to fiber

CSA [21,36].

As regard to postsynaptic factors, the density of AchR

and the rate of hydrolysis by AChE cannot be neglected. The

density of AChR presented a decreased trend from the DIA to

the SOL and bottomed in the EDL in our experiment. In some

diseases such as burn and denervation, receptor prolifera-

tion, mostly extrajunctional, would increase the density of

AChR. In the pathologic states, density of AChR accounted

for the resistance to NDMR. However, the relationship be-

tween density of AChR and response cannot apply to in

normal states.

The affinity of the AChR was also one reason for the

different response to the muscle relaxant. Different affinity of

the AChR in each type of muscle was demonstrated. The de-

gree of saturation of AChRs increased in a dose-dependent

manner in all three groups of muscles as the concentration

of rocuronium increased. Within each concentration, the in-

crease in the DIA was significantly smaller than that in the

EDL and SOL. Compared with the EDL and SOL, the DIA would

havemuchmore unoccupied receptors to retain the activity of

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 4 7 1e4 7 9478

the neuromuscular transmission sufficient to obtain electro-

physiologic actions. Lower the affinity for rocuronium was

more sensitive than the AChR to muscle relaxants [21]. The

DIA showed the lowest affinity of the AChR, whereas the SOL

showed the highest affinity. The results were consistent with

our pharmacodynamic findings.

By comparing morphologic composition of muscle fibers

and end plates of rat DIA, EDL, and SOL muscles, we observed

the number of AChRs per unit fiber CSA and the affinity of the

AChRs and found that theDIAwas characterized by the largest

number of AChRs per unit fiber CSA and the lowest affinity of

the AChR. Compared with SOL, EDL group was proved to have

larger number of AChRs per unit fiber CSA and the lower af-

finity of the AChR. These findings may be the mechanisms of

the different potency to rocuronium in DIA, EDL, and SOL. The

results of the study could help to get a point at the relationship

between different composition of muscle fibers and the po-

tency to muscle relaxants. Extra caution should be taken in

clinical practice when monitoring for muscle relaxation in

anesthetic management using different muscles.

Acknowledgment

This study is supported by the National Natural Science

Foundation of China (grant number 81171845) and Songjiang

District Foundation of Shanghai (grant number 2011PD13).

Authors’ contributions: L.H., M.Y., L.C., and S.L. conceived

and designed the experiments. L.H. and M.Y. performed the

experiments. L.H. contributed to analysis and interpretation,

data collection, and writing of the article. L.C. and S.L. did the

critical revision of the article. S.L. obtained the funding.

Disclosure

The authors reported no proprietary or commercial interest in

any product mentioned or concept discussed in this article.

The authors declare no conflict of interest.

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