| Background and objectiveLaparoscopic surgery has been performed in patients with good physical health since it was used in abdominal surgery in the 1980s. Compared with traditional open surgical procedures, it can reduce postoperative pain, systemic stress response and complications, shorten hospital stay and recovery time, cut medical costs and provide cosmetological effects. All the advantages mentioned above make a rapid popularization of laparoscopic surgery. Due to laparoscopic technique tending to be more and more mature, laparoscopic surgery brings about minimally invasive incision, correct judgments of acute inflammation and potential ability of immunity to inflammation. Therefore, the range of application of laparoscopy has been expanded and used in treatments of various surgical infectious diseases, such as acute appendicitis, acute cholecystitis, gastrointestinal perforation, renal tuberculosis, etc.Carbon dioxide (CO2) is commonly used for pneumoperitoneum in laparoscopic surgery because it's easily attained, non-combustible, metabolized quickly and chemically stable. In early periods, it was suggested that the weak stimulation in treatments entirely attributed to small incisions. However, more and more evidence indicated that the injection of CO2 plays a key role in reducing local and systemic inflammatory responses. There is controversy whether it's suitable for performing laparoscopy and CO2 pneumoperitoneum in patients undergoing inflammation. Infection causes immune responses; meanwhile the surgical operation and CO2 pneumoperitoneum have some influence on the immune response, including white blood cell count, levels of inflammatory factors, etc. Uncertainty immune and inflammatory responses caused by tissue trauma before and after surgery, which are extremely weak in the conventional surgery, even cannot be observed in conventional laparoscopic surgery.The role of CO2 pneumoperitoneum played in the stress response has not been fully and integrally understood. To date, there is not enough convincing evidence that CO2 pneumoperitoneum increase or decrease infection, which is well worthy to be investigated. At the early stage of inflammation, macrophages undergo activation and release inflammatory factors and attacked and non-selective cytotoxic moleculars, such as free radicals. Free radicals participate in the initial and propagate of a variety of diseases. When a large amount of free radicals form in cells, these free radical molecules attack the cell membranes and cause lipid peroxidation and fatty acids broken, which lead to damages to the structure of cell membranes and changes of membrane permeability, resulting in cell damage. Therefore, eliminating and inhibiting free radicals, as well as improving the resistance of oxidation, are important ways for prevention and treatments of disease.To investigate the mechanism of the effect of CO2 pneumoperitoneum on inflammatory factors, we studied the release of superoxide anion and changes of mitochondrial function in macrophages in vitro under the conditions of CO2 pneumoperitoneum. According to the results, we evaluated the feasibility of CO2 pneumoperitoneum in laparoscopic surgery in the condition of inflammation.Materials and methodsUsing the rat alveolar macrophage cell line NR8383 and Queue Multi-gas incubator (United States), we provided 99% CO2 to interfere with the growth of macrophages, which simulated CO2 pneumoperitoneum environment. Pretreatment of adjustable multi-gas incubator for hypoxic environment before the experiment:the hypoxic gas mixture (99% CO2,1% O2) was inflated through the stomata at the rate of 20ml/min and then the stomata were closed after 30 min. Pressure, temperature and relative humidity were controlled at 15 mmHg,21℃, and 2% respectively.Test 1:The effect of CO2 pneumoperitoneum on superoxide anion in rat alveolar macrophage in condition of inflammation. Chemiluminescence determination of free radicals is one of the most sensitive methods. The principle is that the electron of the product, which is derived in the reactions between chemiluminescence reagents and oxygen free radicals and is in excited state, return to the ground state by the non-radiative transitions and emit photons. We detected the release of free radicals in macrophages by universal microplate spectrophotometer in the condition of simulated CO2 pneumoperitoneum environment and the resumed normal oxygen environment. To explore whether CO2 has special effect on the process, we used zymosan to simulate inflammation environment. Cells cultured with zymosan by conventional methods were used as a positive control of hypoxia model.Test 2:The effect of CO2 pneumoperitoneum on mitochondrial function of rat alveolar macrophages in the condition of inflammation. Mitochondrial is the center of energy metabolism in cell, therefore the changes in mitochondrial function is the direct cause triggered apoptosis cascade in cell. We detected the mitochondrial functional state. Methyl thiazol tetrazolium (MTT) assay assesses the viability status of the cells through rapid and simple color reaction. The principle is that MTT can be used as substrate of succinate dehydrogenase in mitochondria of mammalian cells. Mitochondrial succinate dehydrogenase reduces MTT to an insoluble, dark purple formazan crystals deposited in cells. The crystals can be dissolved by dimethyl sulfoxide (DMSO) and the absorbency is measured at wavelength of 450 nm by enzyme-linked immune detector. The absorbency can reflect indirectly the amount and activity of living cells.ResultsTest 1:There were significant differences (P<0.05) at each time point in experimental control group and in normal control group. Significant differences (P<0.05) existed in CO2 pneumoperitoneum group and in experimental control group at 1 hr,2 hr,4 hr and resumed normal oxygen conditions for 2 hr. There was no significant difference in CO2 pneumoperitoneum group and in experimental control group after restored normal oxygen conditions for 4 hours. There were significant differences (P<0.05) in CO2 pneumoperitoneum group and in normal control group at 1 hr,2 hr,4 hr and resumed normal oxygen conditions for 2 hr,4 hr.Test 2:There were significant differences (P<0.05) at each time point in experimental control group and in normal control group. Significant differences (P<0.05) existed in CO2 pneumoperitoneum group and in experimental control group at 1 hr,2 hr,4 hr and resumed normal oxygen conditions for 2 hr. There was no significant difference in CO2 pneumoperitoneum group and in experimental control group after restored normal oxygen conditions for 4 hours. There were significant differences (P<0.05) in CO2 pneumoperitoneum group and in normal control group at 1 hr,2 hr,4 hr and resumed normal oxygen conditions for 2 hr,4 hr.ConclusionsWe investigated the effect of CO2 pneumoperitoneum on release of superoxide anion and changes of mitochondrial function in macrophages in vitro under the conditions of inflammation. The results indicated that there were significant differences (P<0.05) in CO2 pneumoperitoneum group and in experimental control group at 1 hr,2 hr,4 hr and resumed normal oxygen conditions for 2 hr. No significant difference was found in CO2 pneumoperitoneum group and in experimental control group after restored normal oxygen conditions for 4 hours. Our results implicated that CO2 pneumoperitoneum can inhibit the release of superoxide anion and mitochondrial function in macrophage, and then reduce the release of oxygen free radicals and the local inflammation, ultimately inhibit cell apoptosis. Meanwhile, the inhibition is short-lived and reversible, which is conducive to ensure the defense function of after surgery.Highlights1. The effect on the rat alveolar macrophage cell line NR8383 in simulated CO2 pneumoperitoneum environmen was investigated.2. The feasibility of CO2 pneumoperitoneum in laparoscopic surgery in the condition of inflammation was evaluated by detecting release of superoxide anion and changes of mitochondrial function in macrophages in vitro under the conditions of simulated CO2 pneumoperitoneum.3. The results implicated that CO2 pneumoperitoneum can inhibit the release of superoxide anion and mitochondrial function in macrophage, and then reduce the release of oxygen free radicals and the local inflammatory response, ultimately inhibit cell apoptosis. Meanwhile, the inhibition is short-lived and reversible, which is conducive to ensure the defense function of after surgery.
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