The Role And Mechanism Of Heterogeneous Nicotinic Acetylcholine Receptors In Decreased Sensitivity Of Nondepolarizing Neuromuscular Block Agents To Skeletal Muscle Induced By Sepsis

Object Research on the dose-response and time-response ofvecuronium, a Non-depolarizing neuromuscular blockade agents indifferent stage of Sepsis induced by cecal ligation and puncture (CLP) onthe rats. And to elaborate the mechanism of pharmacological alteration ofvecuronium in Sepsis in terms of heterogenous nAChR on postsynapticmembrane of neuromuscular junction. And to confirm that whether aprotease inhibitor, ulinastatin improve pharmacodynamic effects ofvecuronium on septic rats, and elucidate that the role of ulinastatin onheterogenous nAChR on postsynaptic membrane of neuromuscularjunction.MethodsPartⅠ66adult male SD rats (age:2~3months, weight range:200~220g) were include and weighed daily. All the rats were randomly divided intothree groups:(i) the normal group in which no operation was performed(n=6);(ii) the Sham group in which a Sham operation was performed (n=6);and (ⅲ) the Sepsis group in which caecal ligation and puncture (CLP) wasperformed. Given the time frame of the animal model, the rats in the Shamand Sepsis groups were assigned to four subgroups: Day1(n=6), Day3(n=6), Day7(for Sham group n=6, for Sepsis group n=12), and Day14(for Sham group n=6, for Sepsis group n=12). All the rats were killed, andspecimen collection and detection were performed at the1stday, the3rdday,the7thday and the14thday after CLP/operation respectively.Neuromuscular block was monitored by evoked mechanomyographyusing a nerve stimulator and a force transducer. The tendon of the tibialisanterior muscle was attached to separate transducers. Both sciatic nerveswere exposed at the thigh. Stimulation electrodes were attached to measurenerve-mediated contraction of the tibialis anterior muscle. A preload of30g was applied to yield maximal isometric contractions. The nerve-evokedtensions of the respective tibialis anterior muscles were recorded by anamplifier and displayed using the RM6240USB2.0S (I) version1.0.2software. The isometric twitch tension of the tibialis anterior muscle wasestimated by researchers who were blinded from the experimental group ofeach specimen.Twitch tension was elicited by indirect supramaximal constant current stimulation at0.1Hz using a stimulator and a constant-current unit todeliver twitch tension. All forces were measured in grams. After the elicitedtwitch tension was stabilized, single-twitch tension (averaged in groups offive) was determined. The potency of vecuronium was tested using thecumulative dose–response method. Bolus doses of vecuronium wereadministered i.v. in increments of0.05–1mg/kg until the twitch heightachieved maximal depression. Each incremental dose was given only afterthe twitches were allowed to recover to baseline values. The twitch tensioninhibitory effect was normalized by the following equation:%inhibition=100×(1﹣A/B), where A represents the minimal twitch height inthe presence of vecuronium and B is baseline values of twitch height.Dose-response analysis: inhibitory effect vs dose data was fitted using afour-variable logistic sigmoidal dose-response model. IC50, which is theeffective dose eliciting50%of the maximal effect, was calculated byGraphPad Software.Part Ⅱ60adult male SD rats (age:2~3months, weight range:200~220g)were include and weighed daily. All the rats were randomly divided intotwo groups:(i) the Sham group in which a Sham operation was performed(n=6); and (ⅱ) the Sepsis group in which caecal ligation and puncture(CLP) was performed. Given the time frame of the animal model, the ratsin the Sham group were assigned to four subgroups: Day1(n=6), Day3 (n=6), Day7(n=6), and Day14(n=6). And the rats in the Sepsis groupwere assigned to four subgroups: Day1(n=6), Day3(n=6), Day7(n=12),and Day14(n=12). All the rats were killed, and specimen collection anddetection were performed at the1stday, the3rdday, the7thday and the14thday after CLP/operation respectively.Electromyographic (EMG) recordings were measured at pre andpost-modelling. The data were obtained from the right sciatic nerve thatwas stimulated supramaximally (intensity,3V; duration,0.2ms; andfrequency,1Hz) by a direct stimulation electrode (RM6240Systems, Inc.,Cheng Du, China) from the sciatic nerve. Compound muscle actionpotential (CMAP) was recorded using a superficial disc electrode locatedon the tibialis anterior muscle before and at different times after surgery, asdescribed previously. Electromyographical analysis used theRM6240USB2.0S (I) version1.0.2software (RM6240Systems, ChengduInstrument Company, Chengdu, China), with amplitude, and duration ofCMAP as the parameters. The motor conduction velocity (MCV) wascalculated as the distance of conduction/latency time. The temperature ofeach rat was kept at~36–37℃using a heating light. Neuromusculardysfunction was defined as a decrease of≥20%of the lower limit of thenormal CMAP amplitude.Expression of fetal (γ-nAChR) and neuronal type7-nAChR on thetibialis anterior muscle was assessed using Immunofluorescence and western-blotting. The mRNA encoding for γ-and7-subunits wasevaluated by real-time polymerase chain reaction. And expression ofneuregulin-1of motor neurons in spinal dorsal horn using western-blotting.Part Ⅲ24adult male SD rats (age:2~3months, weight range:200~220g)were include and weighed daily. All the rats were randomly divided intotwo groups:(i) the0.9%N.S group (n=12); and (ⅱ) the ulinastatin group(n=12). All the rats were undergoing CLP. After10days of CLP, the rats inthe Sepsis group were i.v. injected with ulinastatin (5000U/kg)(ulinastatingroup) or0.9%normal saline (0.9%N.S group).Pharmacodynamics of vecuronium was detected using the samemethod as PartⅠ. And EMG recordings and expression of γ-nAChR and7-nAChR was measured as methods in PartⅡ.ResultPartⅠ（1）Vecuronium reduced the magnitudes of indirectly elicited twitchtensions in a dose-dependent manner (P<0.01). Fitting the vecuroniumdose–response data to the four variable logistic equation yielded IC50values, which quantitatively indicate the position of the curve. Comparedwith Normal group, there is no difference between each group in Shamgroup, but significant difference was been found in each group of Sepsis group. The IC50of vecuronium gradually increased from Day1to Day14in the Sepsis group (P<0.01). The IC50ratio, which standardizes themagnitudes of the rightward shifts of the dose–responses curves, wassignificantly the largest for the rats in Day14group of Sepsis group,second largest for the rats in the Day7group, and smallest on Days1and3in the Sepsis group (P<0.01). The slope at IC50, which characterizes theslope of the curves, was not significantly different between the Sham andDay1in the Sepsis group (P<0.05). In the Sepsis group, the slope at IC50gradually increased from Day1to Day14in the Sepsis group (P<0.01).（2） Compared onset time of vecuronium in Sham group with that inNormal group，there is no significant differences (P>0.05)。Compared witheach group in Sham group, there is no significant differences（P>0.05）。InSepsis group, the onset time of vecuronium in Day1group compares withthat of Normal group, there is no significant differences (P>0.05) and theother groups compares with Normal group，there is significant differences（P<0.05）。Compared with the same time subgroups, there is nosignificant differences in Day1group between Sepsis and Sham groups（P>0.05）and there is significant differences between the other groups(P<0.05).Compared clinical duration time of vecuronium in Sham group withthat in Normal group，there is no significant differences (P>0.05)。Compared with each group in Sham group, there is no significant differences（P>0.05）。In Sepsis group, the clinical duration time ofvecuronium in Day1group compares with that of Normal group, there isno significant differences (P>0.05) and the other groups compares withNormal group，there is significant difference（sP<0.05）。Compared with thesame time subgroups, there is no significant differences in Day1groupbetween Sepsis and Sham groups （P>0.05） and there is significantdifferences between the other groups (P<0.05).Compared recovery time of vecuronium in Sham group with that inNormal group，there is no significant differences (P>0.05)。Compared witheach group in Sham group, there is no significant differences（P>0.05）。InSepsis group, the recovery time of vecuronium in Day1group compareswith that of Normal group, there is no significant differences (P>0.05) andthe other groups compares with Normal group, there is significantdifferences (P<0.05). Compared with the same time subgroups, there is nosignificant differences in Day1group between Sepsis and Sham groups(P>0.05) and there is significant differences between the other groups(P<0.05).PartⅡ（1）In the Sepsis group, the amplitudes of CMAP decreasedsignificantly, and the peak appeared on Day14postoperation (P<0.01). Theduration of CMAP in the Sepsis group was prolonged significantly, and thepeak appeared on Day14post-operation (P<0.01). The nerve conduction velocity significantly decreased in the Sepsis group compared with that inthe Sham group (P<0.01). The peak appeared on Day14. In the Sepsisgroup, the twitch tension magnitudes elicited by continuous stimulationswere the largest in the Day1group, second largest in the Day7group, andsmallest in the Day14group (P<0.05).（2）In the Sham muscle specimens, γ-and7-nAChR stains were notobserved in the skeletal muscle membrane. In the septic muscle specimens,the skeletal muscle membrane showed γ-and7-nAChR immunoreactivityfrom Day1to Day14. On Day14, the number of γ-and7-nAChR on theskeletal muscle membrane peaked by positive immunostaining withanti-nAChR γ-antibody and anti-nAChR7antibody. The relative proteinlevels to GAPDH of γ-and7-nAChR significantly increased on Day1,3,7, and14in the Sepsis group compared with those in the Sham group. Thepeak value appeared on Day7and14post-procedure for γ-and7-nAChR,respectively, by semi-quantitative analysis (P<0.05). The γ-and7-subunits of mRNA were detected in all muscle samples by RT-PCR.Similar to the results of western blot, the ratio of γ-and7-mRNA toGAPDH mRNA significantly increased on Days1,3,7, and14in theSepsis group compared with that in the Sham group. The peak valueappeared on Day7and14post-procedure for γ-and7-nAChR,respectively (P<0.05). Statistically significant positive correlations werefound between the IC50of vecuronium and γ-nAchR protein level in the Sepsis group (r=0.864, P=0.026). The level of7-nAchR protein wascorrelated positively with the IC50of vecuronium in the Sepsis group(r=0.765, P=0.002).（5）Compared with Sham group, expression of neuregulin-1decreasedon motor neurons in the spinal dorsal horn of rats in Day1、Day3、Day7and Day14group. At the14thday after CLP, expression ofneuregulin-1was lowest (P<0.05). With the longer annimal modeling,expression of neuregulin-1protein decreased (P<0.05).Part Ⅲ（1）There was a significantly differences in amplitude and MCV ofCMAP between Ulinastatin group and0.9%N.S group. However durationof CMAP have no significantly differences between these group（sP>0.05）.（2） Single twitch tension of tibialis anterior muscle of rats inUlinastatin group was higher than that of rats in0.9%N.S group (P<0.05).（3）On Day14of0.9%N.S group, the relative protein levels toGAPDH of γ-and7-nAChR on the skeletal muscle membrane peaked bypositive immunostaining with anti-nAChR γ antibody and anti-nAChR7antibody. After ulinastatin administration, the expression levels of γ-and7-nAChR decreased in the skeletal muscle membrane on Day14,compared with those of0.9%N.S group (P<0.05).（4）Compared with0.9%N.S group, expression of neuregulin-1 decreased on motor neurons in the spinal dorsal horn of rats in Ulinastatingroup (P<0.05).Conclusions（1）Sepsis attenuates the intensity of the neuromuscular blockingeffect of vecuronium, non-depolarizing neuromuscular blockade agent. Itprolonged the onset time, and reduced the duration and recovery time ofvecuronium. Meanwhile, Sepsis results in neuromuscular dysfunction,twitch tension of skeletal muscle decreased. Amplitude of CMAP and MCVdecreased. Thses indicated Sepsis induced the occurrence of demyelination.（2）Sepsis reduced an up-regulation of expression of γ-nAChR and7-nAChR, and distributed throughout the skeletal muscle cell membrane.It demonstrates that the pharmacodynamics of NMBA changes in Sepsis isassociated with the expression of7-nAChR and γ-nAChR in the skeletalmuscle.（3）The protease inhibitor ulinastatin inhibits the expression ofγ-nAChR and7-nAChR on skeletal musclar of septic rats, and promotethe recovery of neuromuscular function, improve the sensitivity of theNMBA.