ABSTRACT
Objective To investigate the structural response of diaphragm and soleus of the rat after mechanical ventilation (MV), and to explore the specific mechanism of the dysfunction of both muscles.Methods Sixteen male Sprague-Dawley (SD) rats were randomly divided into control group and MV group, with 8 rats in each group. Rats in MV group were treated with controlled ventilation and maintained anesthesia, and those in control group were only anesthetized without MV and maintained anesthesia. The diaphragm and soleus were harvested after MV for 18 hours, and the morphology changes were observed with light microscope. The cross section of muscle fiber was observed by immunofluorescence technique analysis, and the cross-sectional area of muscle fiber was calculated. The ultra structural changes in muscle fibers were observed under transmission electron microscope.Results ① Observed under light microscope, the cross section of the diaphragm and soleus muscle in the control group was regular, the nucleus was normal and the cytoplasm was homogeneous. The fibers in the diaphragm-biopsy specimens from MV subjects were smaller than those from control subjects, whereas these signs were not found in soleus. But fiber atrophy in MV specimens was not accompanied by an inflammatory-cell infiltrate. ② Under the fluorescence microscope, the control group had a smaller cross-section of the slow-twitch muscle in diaphragm, while the fast-twitch muscle fibers werelarger. As compared with diaphragm-biopsy specimens from control, specimens from MV subjects showed decreased cross-sectional areas of slow-twitch and fast-twitch fibers, respectively (μm2: 1069.00±155.24 vs. 1297.12±331.15, 2279.66±442.31 vs. 3031.80±596.11, bothP 0.05). ③ In the control group, the muscle fibers of the diaphragm and soleus were arranged orderly, the boundary of the light and dark bands and the Z-line were clear, and there was no autophagy in the visual field. The outer membrane of the mitochondria was complete, and the cristae were in the shape of clapboard. The signs of misalignment of myofibrils, disruption of Z-line and vacuolar mitochondria were found in diaphragm from MV group, whereas these signs were not found in soleus. Diaphragm from MV group exhibited an increase in autophagic vesicles visualized by transmission electron microscopy as compared with control group.Conclusions Controlled MV for 18 hours resulted in diaphragmatic inactivity and promoted muscle injury and atrophy, while autophagy and mitochondrial dysfunction were enhanced. Soleus immobilization for 18 hours was not associated with muscle atrophy. These facts suggest that the signaling associated with diaphragm atrophy during MV may involve different mechanisms compared with other models of muscle atrophy. Diaphragm appeared to be more susceptible to MV.
ABSTRACT
Objective To investigate the effect of calpeptin on diaphragmatic injury and atrophy under controlled mechanical ventilation in rats.Methods A total of 24 SPF Sprague-Dawley (SD) rats were randomly divided into anesthetized control group (CON group),24-hour controlled mechanical ventilation group (CMV group),and 24-hour CMV + treatment with calpeptin group (CMVC group),with 8 rats in each group.Animals in the CON group received an intraperitoneal injection of pentobarbital sodium without CMV and continuous infusion of pentobarbital sodium.A small-animal ventilator was used for 24 hours in rats of CMV group.Rats of CMVC were treated with a specific calpain inhibitor calpeptin (4 mg/kg).The drug was injected subcutaneously 2 hours before and 8,15 and 23 hours after mechanical ventilation.Changes in diaphragm ultrastructure,light microscopic picture,and myosin heavy chain (MHC) expression were observed.Results ① Alignment of myofilaments and normal Z-band,and the shape of mitochondria were maintained in CON group as revealed by electron microscope.The signs of misalignment of myofibrils,disruption of Z-band and vacuolar mitochondria were found in CMV group,and they were obviously improved in CMVC group.The density of muscle injury (× 10-2/μm2) in CMV group was significantly higher than that in control group (36.8 ± 13.7 vs.6.4 ± 6.3,t=6.373,P=0.001),and that in CMVC group was significantly lowered (17.6 ± 9.1 vs.36.8 ± 13.7,t=3.694,P=0.002).② In CON group,the diaphragm fibers appeared regular in cross section without pathologic change under light microscopy.Fuzzy muscle striations,irregular muscle fibers,centralized nuclei and swelling of capillary endothelial cells were observed in CMV group,while pathological changes in the CMVC group were milder significantly.③ In CMV group,the density of MHCslow and MHCfast was lower compared with that of CON group,and the gray value was lowered by 61.1％ (t=8.138,P=0.001) and 77.1％ (t=8.844,P=0.001),respectively,especially in MHCfast.However,the gray values of MHCslow and MHCfast were increased by 1.51 folds (t=4.601,P=0.010),and 1.33 folds (t=2.859,P=0.011),respectively,after treatment with calpeptin,and the elevation was more significantly in MHCslow.Conclusions Diaphragmatic injury and atrophy were found after CMV for 24 hour.Calpeptin could reverse the detrimental effects of CMV,and it suggested that calpain plays an important role in modulating the ventilator-induced dysfunction of the diaphragm.