Study Of The Effect Of Carbon Dioxide Pneumoperitoneum On Pneumodynamics And Pulmonary Oxidative Stress

Objective:To investigate the effect of carbon dioxide pneumoperitoneum on pneumodynamics and oxidative stress markers level in laparoscopic patients. In this study, we also investigate the effect of carbon dioxide pneumoperitoneum on pneumodynamics and oxidative stress markers level in serum and the lung tissue in pneumoperitoneum rat models, and further examine the effect of Nrf2 on the signal transduction system of carbon dioxide pneumoperitoneum induced oxidative stress in order to prevent perioperative lung injury.Contents and methods:Part 1: Study of the changes of pneumodynamics and markers of oxidative stress in laparoscopic patients following carbon dioxide pneumoperitoneumSixty patients (ASA I-II,20～60y) undergoing elective laparoscopic surgery under general anaesthesia were enrolled in this study and these patients were divided into two groups according to different surgery: patients in group A underwent laparoscopic cholecystectomy and group B underwent retroperitoneoscopy for renal or adrenal surgery. Endotracheal intubation was performed following induction with midazolam 0.06mg/kg, fentanyl 4μg/kg, cisatracurium 0.2mg/kg and the value of BIS under 60. The changes of pneumodynamics parameters were recorded at pre-insufflation(T0), insufflation(T1), insufflation 5 minutes(T2), 15 minutes(T3), 30 minutes(T4), 60 minutes(T5), 120 minutes(T6) and desufflation(T7), desufflation 5 minutes(T8), 15 minutes(T9), 30 minutes(T10). The concentrations of MDA and 8-iso PGF2αin the serum were determined by enzyme-linked immunosorbent assay (ELISA).Part 2: The study of relationship between serum and lung homogenates levels of oxidative stress markers in rat following carbon dioxide pneumoperitoneumFifty-eight male Sprague-Dawley rats weighing 250 to 280g, 3~4 weeks, were used.Ten rats among these were performed tracheotomy and controlled breath using ventilator with tidal volume 0.8～1.0ml/100g and frequency 85 bpm. The changes of pneumodynamics parameters were recorded at pre-insufflation(T0),insufflation(T1),insufflation 5 minutes (T2),10 minutes (T3),15 minutes (T4),30 minutes (T5),60 minutes (T6),90 minutes (T7),120 minutes (T8) and desufflation(T9),desufflation 5 minutes (T10),15 minutes (T11),30 minutes (T12). All rats were allowed to breath spontaneously during the experiments.Other forty- eight rats were randomly divided into two groups, which are sham group (n=6) and pneumoperitoneum group(n=42). In sham-operation group, the Veress needle was inserted into the peritonium without CO2 gas insufflation. In pneumoperitoneum group, rats were randomly divided into eight groups according to different length of pneumoperitoneum. The concentrations of MDA and 8-iso PGF2αin rat serum were measured by ELISA at pre-insufflation(T0),insufflation 30 minutes(T1),60 minutes (T2),120 minutes(T3) and desufflation 30 minutes(T4),2 hours(T5),6 hours (T6) and 12 hours (T7). The concentrations of MDA and 8-iso PGF2αin the lung homogenates were determined by ELISA in each group.Part3: Effects of carbon dioxide pneumoperitoneum on Nrf2 and HO-1 expression in rat lungForty- eight male Sprague-Dawley rats weighing 250 to 280g,3~4 weeks, were randomly divided into two groups, which are sham group(n=6) and pneumoperitoneum group(n=42). In sham-operation group, the Veress needle was inserted into the peritonium without CO2 gas insufflation. In pneumoperitoneum group, rats were randomly divided into eight groups according to different length of pneumoperitoneum. The lungs of rats in each group were removed for examining Nrf2 and HO-1 protein and mRNA expression by realtime RT-PCR and western blot. Differences of Nrf2 and HO-1 expression among groups were compared with LSD test.Results:Part 1: Study of the changes of pneumodynamics and markers of oxidative stress in laparoscopic patients following carbon dioxide pneumoperitoneum1. Pneumodynamics parametersIt had few effect of carbon dioxide pneumoperitoneum on Vt-alv and Mvalv,the value of which were changed at insufflations and desufflation. Cdyn was declined after insufflated, while PIP, ETCO2, VCO2, Raw were increased compared with pre- insufflation time points.The effection of carbon dioxide pneumoperitoneum on them depends on the duration and position of pneumoperitoneum.2. Hemodynamics parameters The changes of hemodynamics parameters during pneumoperitoneum were transient.CO,SV and CI were all decreased with insufflation.3.The serum concentration of MDA and 8-iso PGF2αhad the same tendency in each group.They dramatically increased at insufflations 30 minutes,and increased with the prolonged pneumoperitoneum . Part 2: The study of relationship between pneumodynamics and markers of oxidative stress in rat lung following carbon dioxide pneumoperitoneum1. The changes of pneumodynamics parameters were transient and reversible following carbon dioxide pneumoperitoneum.The value of MV and Cdyn were declined at the beginning of insufflations,meanwhile the value fo dPpl and RI were increased.2. The concentrations of MDA and 8-iso PGF2αin serum were 125.68±25.47 and 75.12±11.35(pg/g)respectively before insufflations. The levels of the markers were increased with prolonged insufflations.The concentrations of MDA and 8-iso PGF2α were the highest in time point after insufflations 2 hours.After desufflation 2 hours,they were declined to the level of pre-insufflation until 12 hours after desufflation.3. The concentrations of MDA and 8-iso PGF2αin in the lung tissue were 266.78±31.47 and 150.35±21.87(pg/g)respectively before insufflations.The tendency of two markers were the same as in serum.4. The concentrations of MDA and 8-iso-PGF2a showed a negative association with Cdyn, and a positive association between MDA, 8-iso PGF2αin and dPpl, RI.Part3: Effects of carbon dioxide pneumoperitoneum on Nrf2 and HO-1 Expression in Rat LungRT-PCR showed that the carbon dioxide pneumoperitoneum enhance the expression of Nrf2 and HO-1 mRNA in other groups compared with sham group .The expression of Nrf2 and HO-1mRNA were significantly increased in group P4 .The expression of Nrf2 and HO-1 were returned to basal level in group P7.Western blot showed the expression of Nrf2 nucleus protein begin with insufflations 30 minutes and returned to pre-insufflation until after desufflation 12 hours.The expression of HO-1 protein also increased with prolonged pneumoperitoneum and returned to pre-insufflation until after desufflation 12 hours. The expression of Nrf2 and HO-1 protein were significantly increased in group P4. Nrf2 and HO-1 protein expression in P4 was1.64 and 3.47 times as much as those in group C.Conclusions:1.The effect of carbon dioxide pneumoperitoneum on pneumodynamics and hemodynamics parameters in laparoscopic patients is transient and reversible. They returned to pre-insufflation condition rapidly after desufflation. Carbon dioxide pneumoperitoneum can cause oxidative stress in laparoscopic patients, the degree of which was related with the length of pneumoperitoneum.2. Carbon dioxide pneumoperitoneum can induce pulmonary oxidative stress in rat. The levels of MDA and 8-iso PGF2αincreased in serum and in lung tissue during pneumoperitoneum. The concentrations of MDA and 8-iso-PGF2a showed a negative association with Cdyn, and a positive association between MDA, 8-iso PGF2αin and dPpl, RI.3. Nrf2 invovled in the lung oxidative stress by carbon dioxide pneumoperitoneum, and activated HO-1. The carbon dioxide pneumoperitoneum increase the expression of Nrf2 and HO-1 mRNA and protein.Thus, Keap1-Nrf2/ARE might be the molecular mechanism of pulmonary oxidative stress by carbon dioxide pneumoperitoneum .