Effect And Mechanism Of Different Frequency Neuromuscular Electrical Stimulation On ICU-acquired Weakness Muscular Atrophy In Mice

Objective: ICU acquired weakness(ICU-AW)is an important factor influencing the prognosis of critically ill patients.Till now,neuromuscular electrical stimulation(NMES)is known to be an important method for the prevention and treatment of ICU-AW.However,the optimal frequency of NMES treatment remains to be explored,and the mechanism of NMES is not completely clear.Therefore,we performed a study using lipopolysaccharide(LPS)to construct an ARDS related ICU-AW mice model by intratracheal infusion,and the prevention and treatment effects and mechanisms of NMES at different frequencies on muscle atrophy in ICU-AW mice model were investigated.Methods: Ninety-six healthy male C57BL/6 mice were randomly divided into 12 groups with 8 mice in each group.The specific groups were as follows: blank control group(Control-1,C1),exposed trachea group(Control-2,C2),endotracheal injection of sterile water group(control-3,C3),ICU-AW model group(ICU-AW).After constructing ICU-AW mice model,ICU-AW were divided into two groups to explore the role of AMPK in the pathogenesis of ICU-AW: ICU-AW+AMPK agonist A-769662 group(ICU-AW-A)and ICU-AW+A-769662 solvent group(ICU-AW-V).To study the optimal treating frequency of NMES,ICU-AW model animals were divided into: NMES 20 Hz group(ICU-AW-20),NMES 40 Hz group(ICU-AW-40),NMES 60 Hz group(ICU-AW-60),NMES 80 Hz group(ICU-AW-80).After obtaining information of the optimal treating frequency,ICU-AW model animals were divided into two groups in order to further explore the role of AMPK in NMES: ICU-AW-40+AMPK inhibitor Compound C group(ICU-AW-40-C)and ICU-AW-40+Compound C solvent group(ICU-AW-40-V).Mice in each group were treated as follows:C1: Blank control.C2: The neck skin of the mice was cut to expose the trachea and then the skin was sutured.C3: The neck skin of the mice was cut and the trachea was exposed.Sterile water equal to LPS solution amount was injected into the trachea and then the skin was sutured.ICU-AW: The neck skin of the mice was cut and the trachea was exposed.LPS(3 μg/g)was injected into the trachea and then the neck skin was sutured.ICU-AW-A:Based on ICU-AW mice model,AMPK agonist A-769662(30 mg/kg)was intraperitoneally injected 30 min before LPS injection.ICU-AW-V: Based on ICU AW model,dimethyl sulfoxide(DMSO)of AMPK agonist A-769662 solvent was injected intraperitoneally 30 min before LPS injection.ICU-AW-20: ICU-AW model was given NMES 20 Hz intervention.ICU-AW-40: ICU-AW model was given NMES 40 Hz intervention.ICU-AW-60: ICU-AW model was given NMES 60 Hz intervention.ICU-AW-80: ICU-AW model was given NMES 80 Hz intervention.ICU AW-40-C: Based on ICU-AW mice model,AMPK inhibitor Compound C(20mg/kg)was intraperitoneally injected 30 min before LPS injection,and NMES 40 Hz intervention was performed.ICU-AW-40-V: Based on ICU-AW model,DMSO of AMPK inhibitor Compound C solvent was intraperitoneally injected 30 min before LPS injection,and NMES 40 Hz intervention was performed.After 7 days of treatment,the mice gastrocnemius specimens were collected,and light microscope was applied to observe the pathological changes of the muscle.Western blot and q RT-PCR were used to detect atrogin-1,MURF-1 and AMPK protein and m RNA expressions in mice gastrocnemius muscle.Results: 1.Morphological changes of gastrocnemius muscle,grasping power of limbs,survival rate,Atrogin-1 and Mu RF-1 gene and protein expression of experimental mice in each group: Compared with C1,C2 and C3 groups,the mice in ICU-AW group showed gastrocnemius atrophy,and the grasping strength and survival rate of limbs were significantly decreased(P<0.05),and the expression of Mu RF-1 and Atrogin-1 genes and proteins in gastrocnemius tissues were significantly decreased(P<0.001).2.Expression of p-AMPK protein in gastrocnemius of mice in C3 group and ICU-AW group,morphological changes of gastrocnemius,grasping strength of limbs,Atrogin-1 and Mu RF-1 gene and protein expression of mice in ICU-AW-V group and ICU-AW-A group: Compared with C3 group,p-AMPK protein level in ICU-AW group was significantly decreased(P<0.01);Compared with ICU-AW-V group,muscle atrophy of gastrocnemius was improved,grasping ability of limbs was significantly increased(P< 0.05 or 0.01),Atrogin-1 and Mu RF-1 protein and gene expression were significantly decreased in ICU-AW-A group(P<0.01).3.Morphological changes of gastrocnemius muscle,grasping strength of limbs,Atrogin-1 and Mu RF-1gene and protein expression of mice in each group: Compared with the other four groups,gastrocnemius muscle atrophy of mice in ICU-AWW-40 group was significantly improved;ICU-AW-20 group,ICU-AW-60 group and ICU-AW-40 group had significant improvement in grasping ability of extremities,compared with ICU-AW group(P<0.05),and ICU-AW-40 group had the most significant improvement(P<0.05).The protein and gene expression levels of Atrogin-1 and Mu RF-1 in ICU-AW-40 group were significantly lower than those in other four groups(P < 0.01).4.Morphological changes of gastrocnemius,grasping strength of limbs,Atrogin-1 and Mu RF-1 gene and protein expression in experimental mice in each group: Compared with ICU-AW-40-V group,muscle fibers of ICU-AW-40-C gastrocnemius were significantly atrophied,while grasping strength of limbs was significantly decreased(P < 0.05).The m RNA and protein expressions of Atrogin-1 and Mu RF-1 in ICU-AW-40-C group were significantly higher than those in ICU-AW-40-V group(P < 0.01).Conclusion: 1.An ARDS related ICU-AW mice model could be constructed by intratracheal infusion of LPS.2.AMPK may play an important role in the pathogenesis of ICU-AW mice model.3.40 Hz NMES intervention can significantly improve skeletal muscle atrophy in ICU-AW mice animal model.4.NMES may play a protective role in improving ICU-AW skeletal muscle atrophy by enhancing AMPK activity.