An Experimental Study On Diaphragmatic Function In Rats With Chronic Obstructive Pulmonary Disease

Objectives: Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation that is not fully reversible. The airflow is limited to be usually both progressive and associated with harmful gas or noxious particulate induced abnormality inflammatory reaction in lung. The persistence decreasing of pulmonary function causes damage of patient's work ability and quality of life seriously. Lung is the chief organ that be involved in the patient of COPD. Moreover, Other organs infaust effect can be caused all over the body. It is discovered that skeletal muscle malnutrition, atrophy and somewhat systemic inflammatory reaction all over the body in patient with COPD occur due to hypoxia, acid-intoxication, nutritional disturbance and emphysema. Respiratory muscles like power pump during breath. Diaphragm is the supreme respiratory muscle,to which, it contribute about 60%-80% in respiratory movement. Different dysfunctional level of diaphragmatic muscle were discovered in patient with COPD, especially in acute exacerbation of COPD,in which diaphragm fatigue is significant. Diaphragm fatigue is an important factor that induce respiratory failure. Mechanical ventilation is an important measure to remedy severe respiratory failure. However, inappropriate duration of ventilation and mode of mechanical ventilation can impact diaphragmatic function, even induce difficulty in weaning mechanical ventilation. Therefore, to evaluate diaphragmatic function and treatment diaphragm fatigue appropriately and to use breathing machine correctly are very important to treat respiratory failure in patient with COPD. There are many question need to be explored.Rats with COPD were treated as research objects in this study. To explore diaphragmatic function and the influence of controlled mechanical ventilation on diaphragmatic function in COPD model rats. We applied dripping lipopolysaccharide(LPS)to trachea and passive smoking to replicate model of chronic obstructive pulmonary disease of human. After that, pulmonary function were measured in normal rats and COPD model rats. Trans-diaphragmatic pressure were determined by measuring intra-thoracic pressure and intra-abdominal pressure in all rats. Then electromyogram of diaphragmatic muscle were measured in rats. The change of diaphragmatic function would be evaluated in rat with chronic obstructive pulmonary disease and normal rats. Diaphragmatic function would be observed on rats with COPD after short term controlled mechanical ventilation (6 hours) by determining trans-diaphragmatic pressure and electromyogram of diaphragmatic muscle. In this way, to explore the influence of short term controlled mechanical ventilation on diaphragmatic function. Methods: The SD rats were divided randomly into two groups : A normal control group(n=12), B replicating model group(n=24), the B group rats were divided into two subgroups randomly, the subgroup B1(n=12)were replicated model only, the subgroup B2(n=12)with short term controlled mechanical ventilation after replicating model in term of experiment.The procedure of rats for replicating model of chronic obstructive pulmonary diseases: on the first day and the 14th day, LPS(200ug/200ul) was instilled into the trachea of rats of B group, from second day to 13th day and 15th to 28th day, the rats of B group would be given passively smoking in box (40*50*60cm) twice daily with every time continue for 60 minutes. The rats of control group would be living normally. All rats were breaded in mouse cages and feed intake freely.After duplicating model, rats were given intraperitoneal injection anesthesia of 1% pentobarbital sodium (50mg/kg) and were kept moderately Drugged state. Then cervical part skin of rat were medisected, and trachealed intubation through exposured trachea by blunt dissection architecture. Made a cut (about 1.5cm ) at the center of abdomen to the right near the appendix ensiformis in rats. A myoelectricity electrode was put to the right bony part of diaphragmatic through the cut of abdomen in order to collect the electromyographic signal of diaphragmatic muscle. The signal would be stored in computer. After that, one pipe was inserted to inferior segment of esophagus of rats to measure intra-thoracic pressure, a balloon catheter was placed below the diaphragmatic muscle to collect intra-abdominal pressure. Two pipes were connected with potentiometric transducer and physiological functions test system and computer. Trans-diaphragmatic pressure was measure by this way. At last, rats were put in breathing machine to determine pulmonary function when breathe stable. Pulmonary function parameter include forced vital capacity (FVC) and forced expiratory volume in zero point three second and peak expiratory flow and air way resistance and dynamic compliance of lung. Rats in B2 group were determined trans-diaphragmatic pressure and electromyogram of diaphragmatic muscle before and after mechanical ventilation. In course of experiment, the rats were maintained anesthesia state.After all indexes were determined , rats would be executed. The nether section of the right lung was be put in 10% formaldehyde liquid , and stained with hematoxylin-eosin. The right upper lobe (lung) was stored for other use. Bronchus principalis dexter was ligatured by thread and bronchoalveloar lavage fluid (BALF) was obtained by bronchoalveloar lavage with 0.9% saline in left lung. BALF was collected and centrifugated (1200rpm, 4℃, 10min). Supernatant fluid was stored in -70℃until processing.Results: (1) The light microscope result showed that airway epithelial were intact, ciliary's arrangements was regular and the structure of alveoli was normal in A group. It was manifested that alveoli of lung were different in size, part of them coalesced and expanded in group B. The arteriole smooth muscle in pulmonalis were thickened and lumens were shrinked secretion was seen multiplication in bronchus and cilium of epithelium mucosae were derangement. Beaker cell were increased and smooth muscle under membrane mucosa of bronchus were thickened. All these changes consistent with the characteristic of chronic obstructive pulmonary diseases in human. The pathology morphology showed that muscle fiber arrangement were regular and cross section showed muscle fiber were coherent in diaphragm of normal rats. It was shown that the muscle fibers were not concordant in size and connective tissue and cells between muscle were increased. The light microscope result showed that group B1 and B2 are no evident difference in disphragmatic muscle. (2) Result of pulmonary function: FEV0.3/FVC decreased in rats in chronic obstructive pulmonary disease in model group (P<0.05). The air way resistance were increased and pulmonary compliance were decreased in model group (P<0.05). (3) In the group B the total leukocytes, PMN numbers were higher than those of group A (P<0.05). Comparing to group A, the percentage of macrophage decreased in group B (P<0.05). Percentage of leukomonocyte decreased in group B, but no significant different. (4) Changes of trans-diaphragmatic pressure in model group and control group The transdiaphragmatic pressure of model group (4.28±1.03cmH2O) were lower than control group (5.36±0.94cmH2O) (P<0.05). (5) Results of spectral decomposition of electromyographic signal of diaphragmatic muscle in ratio by high frequency and low frequency were lower in model group (P<0.05) than control group(P<0.05). The change in high frequency was no statistically significant. However it was increased significantly in low frequency in model group. (6) Results of trans-diaphragmatic pressure and spectral decomposition; spectrum analysis of electromyographic signal of diaphragmatic muscle before and after mechanical ventilation in rats of chronic obstructive pulmonary disease model were no significant difference(P>0.05).Statistics: Data are expressed as means±SEM. The differences were analyzed by group t-test. The SAS 6.12 software were used to deal with data. P<0.05 was considered statistically significant different.Conclusion: (1) The rats in the model group by instilling LPS into the trachea combine passive smoking was characterized with emphysema and limit of air flow which indicated the succeed in replicating COPD model. (2) Trans-diaphragmatic pressure and spectrum analysis of electromyographic signal of diaphragmatic muscle are indicated that diaphragmatic function decrease in rats with COPD. Electromyogram is sensitive to detect respiratory muscle fatigue. (3) Short term (6 hours ) controlled mechanical ventilation impact on diaphragmatic function is no significant difference in rats with COPD.