Objective To explore the effects of mechanical ventilation and mechanical ventilation com-bined with phrenic nerve electrical stimulation on the morphology of the phrenic nerve-diaphragm neuromuscular junction in rats.Methods The rats were divided into a control group(CON group,n=6);an 18-hour mechanical ventilation group(MV group,n=6);an 18-hour ventilation combined with sham electrical stimulation group(S-MS group,n=6);and an 18-hour ventilation combined with electrical stimulation group(MS group,n=6),in which the diaphragm contraction force was detected through the biological signal acquisition system,the stimula-tion frequency-contraction curve was fitted,and the muscle fatigue index was calculated.The muscle fiber cross-sectional area(CSA)of the diaphragm specimen was observed through HE staining;the endplate membrane area of the neuromuscular junction(NMJ)was observed through immunohistochemistry(IHC)staining of the diaphragm combined with laser confocal technology.Results As compared with the CON group,the contractility of isolated diaphragm muscles in the MV group and S-MS group decreased;the fatigue resistance of isolated diaphragm,as compared with the CON group(0.686 8±0.049 42),decreased in the MV group(0.360 3±0.066 89)and the S-MS group(0.367 9±0.034 94),with a greater decline in the MS group(0.536 2±0.054 38);as compared with the CON group[674.3(523.9,863.6)],diaphragm CSA was reduced in the MV group[374.5(293.3,522.7)]and S-MS group[392.8(309.5,542.6)],with a relatively lower reduction in the MS group[592.7(425.1,820.0)];as compared with the CON group[393.7(293.5,471.3)],the diaphragm endplate membrane area decreased in the MV group[289.7(227.0,354.2)]and S-MS group[243.1(190.8,331.7)],with a lower degree of reduction in the MS group[331.0(262.8,413.7).Conclusions Mechanical ventilation causes diaphragm dysfunction in rats and decreases the endplate membrane area of the diaphragm NMJ.Bilateral phrenic nerve electrical stimulation for 10 min/h can improve diaphragm dysfunction and changes in the endplate membrane area of the diaphragm NMJ caused by mechanical ventilation.

OBJECTIVE: To explore the role of endoplasmic reticulum ryanodine receptor 1 (RyR1) expression and phosphorylation in sepsis- induced diaphragm dysfunction. METHODS: Thirty SPF male SD rats were randomized equally into 5 groups, including a sham-operated group, 3 sepsis model groups observed at 6, 12, or 24 h following cecal ligation and perforation (CLP; CLP-6h, CLP-12h, and CLP-24h groups, respectively), and a CLP-24h group with a single intraperitoneal injection of KN- 93 immediately after the operation (CLP-24h+KN-93 group). At the indicated time points, diaphragm samples were collected for measurement of compound muscle action potential (CMAP), fatigue index of the isolated diaphragm and fitted frequencycontraction curves. The protein expression levels of CaMK Ⅱ, RyR1 and P-RyR1 in the diaphragm were detected using Western blotting. RESULTS: In the rat models of sepsis, the amplitude of diaphragm CMAP decreased and its duration increased with time following CLP, and the changes were the most obvious at 24 h and significantly attenuated by KN-93 treatment (P < 0.05). The diaphragm fatigue index increased progressively following CLP (P < 0.05) irrespective of KN- 93 treatment (P>0.05). The frequency-contraction curve of the diaphragm muscle decreased progressively following CLP, and was significantly lower in CLP-24 h group than in CLP-24 h+KN-93 group (P < 0.05). Compared with that in the sham-operated group, RyR1 expression level in the diaphragm was significantly lowered at 24 h (P < 0.05) but not at 6 or 12 following CLP, irrespective of KN-93 treatment; The expression level of P-RyR1 increased gradually with time after CLP, and was significantly lowered by KN-93 treatment at 24 h following CLP (P < 0.05). The expression level of CaMKⅡ increased significantly at 24 h following CLP, and was obviously lowered by KN-93 treatment (P < 0.05). CONCLUSION Sepsis causes diaphragmatic dysfunction by enhancing CaMK Ⅱ expression and RyR1 receptor phosphorylation in the endoplasmic reticulum of the diaphragm.
Rats ; Male ; Animals ; Diaphragm/metabolism* ; Ryanodine Receptor Calcium Release Channel/metabolism* ; Rats, Sprague-Dawley ; Phosphorylation ; Muscle Contraction/physiology* ; Endoplasmic Reticulum ; Sepsis/metabolism*