| Chronic obstructive pulmonary disease (COPD) is a common, prevalent pulmonary disease. According to statistics, COPD is a considered to be the fourth leading cause of death. In more than fifteen populations, prevalence rates of COPD reached up to 3% in our country and there are approximately 2500 million COPD patient[1]. Till now, effective therapy for it is unavailable.The diaphragm is the most important inspiratory muscle. Function of diaphragm was 60-80 percent of all respiratory muscle[2].Functional impairment of diaphragmatic muscle is one of the pathological hallmarks of COPD. COPD patients frequently develop diaphragmatic muscle fatigue to result in respiratory impairment. Diaphragmatic muscle fatigue may be prevented and treated by medications for ameliorating diaphragmatic muscle contractility, mechanical ventilation to help diaphragmatic muscle rest, respiratory muscle exercise, and diaphragmatic muscle pacing [3-4]. Chronic electrical stimulation to diaphragmatic muscle was played a important role in the rehabilitation care of diaphragmatic muscle of COPD patients, because it can enhance respiratory muscle contraction and improve tolerance and force[5]. Now, chronic electric stimulation (CES) using physiologic frequency (40Hz) has exhibited certain therapeutic role in the rehabilitation care of diaphragmatic muscle of COPD patients.The use of diaphragmatic muscle pacing is based on the experimental evidence that diaphragmatic muscle, like skeletal muscles, undergoes adaptive changes under electric stimulation. Electric stimulation using various frequencies may cause varying changes in the structure, metabolism and function of skeletal muscles. Diaphragmatic muscle, being skeletal muscle, may undergo adaptive changes similar to those changes of skeletal muscles under chronic electric stimulation.Recent reports indicate [6-7] that patients with mild to moderate COPD present atrophy in type I and type II diaphragmatic muscle fibers, and that patients with severe COPD manifest increases in the percentage of type I fibers, impairment in the contractile function of diaphragmatic muscle, increase in protein degradation, and reduction in Ca2+ sensitivity and periodic locomotion of cross-bridges. Chronic electric stimulation using physiologic frequency may recruit motor units of atrophic diaphragmatic muscle, enhance the function of various types of muscle fibers, and maintain relatively normal percentages of various types of muscle fibers. Hence, physiologic frequency electric stimulation transforms the type of diaphragmatic muscle fibers in COPD patients, but does not lead to optimal muscle fiber remodeling which is required in clinical perspective, and 40Hz is not the optimal frequency for the rehabilitation care of diaphragmatic muscle of COPD patients.Chronic low-frequency electrical stimulation (10Hz) may transform fast fibers of skeletal muscle into slow fibers, a process accompanied by changes in the synthesis of corresponding myosin heavy chain (MHC) isoform and induced adaptative changes in diaphragmatic muscle Ca2+ regulation system and metabolic enzyme activity.That result in increased tolerance of muscle to fatigue and shorten of fatigue recovery. Chronic physiologic frequency electric stimulation can improve functional of atrophic diaphragmatic muscle, enhance the function of various types of muscle fibers. Chronic electric stimulation using both low and physiologic frequencies combine together dominance of two type pattern of electric stimulation and not only enhance respiratory muscle contraction but also increase tolerance, and thus ameliorate respiratory muscle function and the quality of life of COPD patients.In this study, the features and patterns of adaptive changes in diaphragmatic muscle structure, energy metabolism, muscle strip mechanics and E-C coupling was observed in rabbit models of emphysema subjected to electric stimulation using various frequencies. The aim of which was to elucidate the mechanism by which chronic electric stimulation using low and physiologic frequencies (10+40Hz) prevents and treats diaphragmatic muscle fatigue and to provide theoretical basis for the use of in vitro diaphragmatic muscle pacing using low and physiologic frequencies to prevent and treat diaphragmatic muscle fatigue in COPD patients.The main contents of this study are as follows:(I) Establishment of rabbit models of emphysema and animal models of chronic electric stimulation1. Rabbit models of emphysema were established by caroid aerosol inhalation.2. Animal models of chronic electric stimulation were established by using the portable, frequency-adjustable, in vitro diaphragmatic muscle pacemaker developed by the author(s).(II) Experimental observation on changes in MHC and metabolic enzyme activity of diaphragmatic muscle of rabbit models of emphysema following chronic electric stimulation using various frequencies1. Changes in arterial blood gas.2. Changes in diaphragmatic muscle MHC subtypes (assayed by SDS-polyacrylamide gel electrophoresis).3. Changes in the activity of metabolic enzymes (assayed by using the succinate dehydrogenase (SDH) kit and the lactate dehydrogenase (LDH) kit manufactured by Nanjing Jiancheng Bioengineering Institute).(III) Experimental observation on changes in mechanics, calcium release units and Ca2+ uptake and release by and sarcoplasmic reticulum (SR) of diaphragmatic muscle of rabbit models of emphysema following chronic electric stimulation using various frequencies.1. Changes in the mechanic properties of diaphragmatic muscle (assayed by using the Rm6240 multi-channel physiological signal collecting and processing system).2. Changes in diaphragmatic muscle Ca2+-ATPase activity (assayed by using the Ca2+-ATPase kit manufactured by Nanjing Jiancheng Bioengineering Institute).3. Changes in Ca2+ uptake-release kinetics by diaphragmatic muscle SR (assayed by Fura-2 fluorophotometry).4. Effect of chronic electric stimulation using various frequencies on the expression of ryanodine receptor (RyR1-3) mRNA (assayed by RT-PCR).Main results1. Pathological study demonstrated characteristic manifestations of emphysema in rabbit models such as sparse lung tissue, thinning of alveolar septum, compression of capillaries in alveolar septum, disruption and loss of some alveolar septa, and alveolar fusion-induced bullae etc. 2. Following chronic electric stimulation using 40Hz, 10Hz, and 10+40Hz, PaO2 and SaO2 increased, and PaCO2 decreased in rabbits with emphysema (P<0.05). The increase in PaO2 and SaO2 was more significant in the 10+40Hz group than in the 40Hz and 10Hz groups (P<0.05).3. Following chronic electric stimulation using 40Hz, 10Hz, and 10+40Hz, the relative contents of MHC were increased in diaphragmatic muscle of rabbits with emphysema. The percentage of MHC-I was significantly increased in the 10Hz group, while that of MHC-IIa was significantly increased in the 10+40Hz group.4. Following chronic electric stimulation using 40Hz, 10Hz, and 10+40Hz, SDH activity was increased in diaphragmatic muscle of rabbits with emphysema (P<0.05), while LDH activity did not change significantly. SDH activity was increased more significantly in the 10+40Hz group than in the 40Hz and 10Hz groups (P<0.05).5. Following chronic electric stimulation using 40Hz and 10+40Hz, diaphragmatic muscle twitch tension (Pt) and titanic tension (Po) were significantly increased in rabbits with emphysema, time-to-peak tension(TPT) and half-relaxation time (1/2Rt) were significantly reduced, and fatigue index (FI)and fatigue recovery index (FRI) were significantly decreased (P<0.01). In the 10Hz group, Pt and Po were reduced (P<0.05), TPT and 1/2Rt increased, and FI and FRI decreased (P<0.01). The decrease of FRI and FRI was more significant in the 10+40Hz group than in the 40Hz group (P<0.01).6. Following chronic electric stimulation using 40Hz and 10+40Hz, diaphragmatic muscle SR Ca2+-ATPase activity was increased in rabbits with emphysema(P<0.01), and Ca2+ release and uptake rate of SR was significantly increased(P<0.01). RyR1mRNA and RyR3mRNA expression were significantly upregulated(P<0.01). In the 10Hz group, Ca2+-ATPase activity was lowered, and Ca2+ release and uptake rate of SR was significantly decreased (P<0.01). RyR1mRNA expression was down-regulated (P<0.05). Mild RyR2 mRNA expression was observed in the emphysema group and the 10Hz electric stimulation group, but not in other groups.Conclusions1. Chronic electric stimulation using low and physiologic frequencies may transform typeâ…¡b fibers of diaphragmatic muscle of rabbits with emphysema into typeâ… ,â…¡a fibers, the adaptive changes in structure of diaphragmatic muscle is the foundation of changes in function.2. Chronic electric stimulation using low and physiologic frequencies increases the activity of enzymes responsible for oxidative metabolism and aerobic oxidation in diaphragmatic muscle of rabbits with emphysema more significantly than chronic electric stimulation using physiologic frequencies or low frequencies does.3. Chronic electric stimulation using low and physiologic frequencies and chronic electric stimulation using physiologic frequency enhance Ca2+ uptake and release of SR in rabbits with emphysema, improve E-C coupling, increase diaphragmatic muscle inotropy and the fatigue tolerance of diaphragmatic muscle. But following chronic electric stimulation using low and physiologic frequencies the amelioration of respiratory function and atigue tolerance of diaphragmatic muscle are more significant than the effect that chronic electric stimulation using physiologic frequency. It indicate that chronic electric stimulation using low and physiologic frequencies may be the optimal frequency mode for the rehabilitation care of diaphragmatic muscle of COPD patients with extraorgan diaphragm pacing. |
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