Effects Of Midazolam,Dexmedetomidine And Propofol On The Diaphragm Function In Mechanical Ventilated Rats

Background Patients experienced prolonged mechanical ventilation(MV)always accompanied with the loss of diaphragmatic strength and(or)myofibers atrophy,which termed as ventilator induced diaphragm dysfunction(VIDD).Appropriate sedation is also essential during MV,and anesthetics may have direct adverse effects on the diaphragm.It has been reported that propofol itself as an independent factor contributed to the diaphragmatic contractile reduction and muscle fiber atrophy in spontaneously breathed rats.Dexmedetomidine impaired diaphragmatic function but not aggravated diaphragmatic myofibers atrophy in mechanically ventilated rats.Propofol or dexmedetomidine,however,which one owns the minimal influence on the diaphragm under MV is still unclear.Moreover,the effect of midazolam(a classical sedative)on the diaphragm function under MV has not been fully reported.Together,we aimed to compare the effect of midazolam,dexmedetomidine,and propofol on diaphragm function during MV in rats.Objective(1)To compare the effect of midazolam,dexmedetomidine,and propofol on diaphragmatic contractile function and myofibers cross-sectional area(CSA)during MV.(2)To explore the role of oxidative stress,autophagy,diaphragmatic microcirculation,protein synthesis and degradation playing in the diaphragm dysfunction under midazolam,dexmedetomidine,and propofol sedation during MV.Methods(1)Effect of midazolam,dexmedetomidine,and propofol on diaphragm function in spontaneously breathed rats.To explore the direct effect of these sedatives on the diaphragm function,we set a model of rats experienced spontaneous breathing(SB)with midazolam,dexmedetomidine,and propofol sedation for 12 hous.Briefly,rats weighed 300 ± 10 g were randomized assigned into four groups breathed spontaneously with continuous infusion of pentobarbital(PB,n = 6),midazolam(MZ,n = 6),dexmedetomidine(DD,n = 6)or propofol(PP,n = 6).The PB group as the control.Heart rate,mean arterial pressure(MAP)and rectal temperature were continuously monitored.Blood samples were taken every 4 hours for venous blood glucose,arterial blood gas analysis,blood cell counts,electrolytes and lactate examination.Anesthetics dosages were administered to maintain a relatively steady sedation depth,and the sedation depth was evaluated by pedal reflex,corneal reflex,tail reflex,and arterial blood pressure.Twelve hours later,a strip of costal diaphragm muscle and gastrocnemius muscle was dissected for in vitro contractile measurements.Hematoxylin and eosin staining was used for the micro-structural observation,and immunofluorescence staining was performed to assess the CSA of the fibers.(2)Effect of midazolam,dexmedetomidine,and propofol on diaphragm function in mechanically ventilated rats.Rats weighed 300 ± 10 g were randomized assigned into four groups experienced MV with continuous infusion of pentobarbital(PB,n = 5),midazolam(MZ,n = 5),dexmedetomidine(DD,n = 5)or propofol(PP,n = 5)for 12 hours.The PB group as the control.Heart rate,MAP and rectal temperature were continuously monitored.Blood samples were taken every 4 hours for venous blood glucose,arterial blood gas analysis,blood cell counts,electrolytes and lactate examination.The microcirculation of diaphragm and gastrocnemius were measured every 6 hours.Anesthetics dosages were administered to maintain a relatively steady sedation depth,and the sedation depth was evaluated by pedal reflex,corneal reflex,tail reflex,arterial blood pressure,and peak inspiration pressure.Twelve hours later,a strip of costal diaphragm muscle and gastrocnemius muscle was dissected for in vitro contractile measurements.Hematoxylin and eosin staining was used for the micro-structural observation,and immunofluorescence staining was performed to assess the CSA of the fibers.Western-blot was used to measure the diaphragmatic expression of HIF-1?,4-HNE,Catalase,SOD1,SOD2,p-Akt/Akt ratio,Atrogin-1,Murf-1,and LC3B-II/I ratio.The transmission electron microscopy was used for the morphological observation of sarcomere and autophasomes in the diaphragm.Result(1)Effect of midazolam,dexmedetomidine,and propofol on diaphragm function in SB group.At the end of the experiments,MAP was significantly lower in comparison to baseline in all groups.Data of MAP,heart rate,rectal temperature,arterial blood gas,electrolyte,plasma lactate,and blood cell counts before euthanization were not significantly differences in all the SB groups.The maximum tetanic force,force-frequency curve and fatigability test of the diaphragm muscle in the MZ,DD,and PP groups were significantly lower than those of PB group.Differences of these contractile measurements were not significant in the MZ,DD,and PP group.Alterations of the micro-structure in the diaphragm were not observed by HE staining.Meanwhile,CSA of diaphragmatic fibers was also insignificant in all groups.Different from diaphragm,fiber size and contraction force in gastrocnemius were kept unchanged in all groups.Our results revealed that midazolam,dexmedetomidine,and propofol have no effect on the gastrocnemius function under spontaneous breathing condition.However,theses sedatives resulted in the further contraction force reduction without obvious muscle fiber atrophy in the spontaneously breathed diaphragm.(2)Effect of midazolam,dexmedetomidine,and propofol on diaphragm function in MV group.At the end of the experiments,MAP was significantly lower in comparison to baseline in all groups.Data of MAP,heart rate,rectal temperature,arterial blood gas,electrolyte,plasma lactate,and blood cell counts before euthanization were not significantly differences in all the MV groups.The maximum tetanic force,force-frequency curve and fatigability test of the diaphragm muscle in the MZ,DD,and PP groups were significantly lower than those of PB group.MZ group had lower contractile measurements than DD and PP groups.The discriminations of these contractile measurements between DD and PP were not significant.Alterations of the micro-structure in the diaphragm were not observed by HE staining.Immunofluorescence staining showed that the CSA of type II fiber in the MZ group was lower than the DD and PP groups.No significant difference was observed between DD and PP group.Meanwhile,the decline of diaphragmatic functional capillary density was observed in all the groups by sidestream dark filed microscopy.Dexmedetomidine was more efficient in preserving the perfused capillary than midazolam or propofol.Changes of the contractile force,CSA,and microcirculation in the diaphragm wer not observed in the gastrocnemius.The lipid peroxidation adducts 4-HNE and HIF-1? levels were significantly lower in DD and PP groups compared to MZ group.The catalase and SOD1/2 levels were also relative lower(p < 0.05)in MZ group compared to DD or PP group.Meanwhile,the p-Akt/Akt ratio,LC3B-II/I ratio,the expression of Murf-1 and Atrogin-1 in the MZ group were also higher than DD group and(or)PP group.The transmission electron microscopy showed increased autophagosomes in the MZ,DD,and PP groups than the PB group,especially in the MZ and DD groups.In the MZ group,the diaphragm fiber was arranged irregularly,and the Z-line was intermittent.Otherwise,diaphragm fiber was regularly arranged,and the Z-line was distributed continuously in the DD and PP groups.These results suggest that midazolam,dexmedetomidine,and propofol have no adverse effect on the gastrocnemius function under mechanical ventilation.Midazolam,dexmedetomidine,and propofol further deteriorated the diaphragmatic contractile function.However,mechanical ventilation during midazolam sedation led to a more severe diaphragm dysfunction than dexmedetomidine and propofol,possibly caused by its relative weaker antioxidant and anti-proteolytic capacity.Midazolam,dexmedetomidine,and propofol all have the benefit effect in preserving the diaphragm myofibers size from MV;and this effect was more prominent in dexmedetomidine and propofol.Conclusion Midazolam,propofol and dexmedetomidine directly induced similar diaphragmatic dysfunction without obvious muscle atrophy.Midazolam,dexmedetomidine,and propofol further deteriorated the diaphragmatic contractile function.However,twelve hours of mechanical ventilation during midazolam sedation led to a more severe diaphragm dysfunction than dexmedetomidine and propofol,possibly caused by its relative weaker antioxidant and anti-proteolytic capacity.Midazolam,dexmedetomidine,and propofol all have the benefit effect in preserving the diaphragm myofibers size from MV;and this effect was more prominent in dexmedetomidine and propofol.