The Effects Of CO₂ Pneumoperitoneum On Morphology Of Peritoneal Mesothelial Cells And Inflammatory Cytokines In The Abdominal Cavity

BackgroundLaparoscopy has evolved dramatically over the past two decades and become the gold standard for treating a number of digestive and gynecological benign pathologies. Compaired with laparotomy, laparoscopic procedures have been associated with less surgical trauma, less postoperative pain, better esthetic results, reduced hospital stays, and faster recovery. Laparoscopic techniques have been embraced by the public and surgeons alike. Carbon dioxide （CO₂） has been wildiy used during laparoscopic surgery because cheap, colorless, highly soluble and stabilize. However, recent evidence suggests that the use of CO₂ to create a pneumoperitoneum during laparoscopy does not simply create a working space for surgery, but can lead to adverse structural, metabolic, and immune derangements within the peritoneal cavity. Several studies have suggested that CO₂ pneumoperitoneum alters the peritoneal milieu and creates conditions favorable to the invasion and metastasis of tumour cells into the damaged basal membrane. According to Pajet’s‘‘seed and soil’’theory as well as more recent knowledge of cancer biology, tumour cell grow easely at privileged sites. Thus, peritoneum mesothelial cells play an important role in the metastasis and grow of tumour cell within the abdominal cavity.Recently, several studies have examined the relation between pneumoperitoneum and peritoneal trauma. In the scanning electron microscopy study, Volz et al. examined characteristic alterations of the peritoneum after capnoperitoneum, and found condensation of the mesothelial cells, intercellular clefts, and exposition of the basal lamina. This was further supported in another study, however, none of these studies identified the possible cause of this variation.Volz considered the metabolic effects of CO₂ and the distention of abdominal wall were causative factors. Douglas presumed the two major contributors were the increased intraperitoneal pressure （IPP） and the extreme dryness of the gas used rather than the specific gas used for distention. In the event that evaluated and compared the peritoneal ultra-structural alterations caused by CO₂ and air pneumoperitoneum, Marcos concluded that the peritoneal alterations depended on the type of gas. Ordemann presumed gas ?ow to be the in?uence on the integrity of the peritoneum. After observing morphologic alterations of the peritoneum after CO₂, helium and air pneumoperitoneum, Suematsu concluded the alteration was associated with the type of insufflated gas, the amount of pressure, and the duration of pneumoperitoneum. At the same time, Suematsu suggested that peritoneal morphologic alterations after pneumoperitoneum differed from that after laparotomy, but they didn’t demonstrate which was more intensive. Until now, the scientists failed to ascertain the substantial cause of the peritoneal alterations, and the peritoneal morphologic alteration between open and laparoscopic procedures remains unknown at present. On the other hand, without surgical manipulation and respirator support, animal experiment is different from clinic. Thus, the peritoneal morphologic trauma between laparoscopy-assisted gastrectomy and conventional open gastrectomy is still unknown.The surgical trauma to the abdominal cavity cause acute inflammatory reaction of body, which associated with the release of inflammatory factor and growth factor in abdominal cavity. The level of interleukin -1β（IL-1β） in abdominal cavity increase afert surgery and play an important role in inflammatory reaction. Interleukin -6 （IL-6） is also an important inflammatory factor after surgery and the level increase after trauma. IL-1βand IL-6 are early and sensitive markers for analysis of major surgical trauma and the level of them is believed to occur in relation to surgical stress. Previous studies have demonstrated a correlation between CO₂ pneumoperitoneum and inflammatory cytokines in the abdominal cavity, but the effect of different CO₂ pneumoperitoneum on inflammatory factor in abdominal cavity is remained unknown.Therefore, in this study the pneumoperitoneum was established using different type of insufflated gas, the amount of pressure, the duration and gas ?ow, the peritoneum and peritoneal fluid were sampled after pneumoperitoneum, the scanning electron microscope （SEM） and stereology were applicated to estimate the area fraction of the basal lamina exposed and the diameter of the mesothelial cells, ELISA immunoassay were adopted to evaluate the concentration of IL-1βand IL-6. Finally, the result of pneumoperitoneum was compared with that of laparotomy. In order to elucidate the influence in clinical practice, the peritoneum were sampled after Laparoscopy-assisted gastrectomy and conventional open gastrectomy, the SEM and stereology were applicated to estimate the area fraction of the basal lamina exposed and the diameter of the mesothelial cells, the peritoneal morphologic trauma was compared between two groups. Thus, we hope to offer a novel theoretical and experimental basis for reducing the peritoneal trauma and improving clinical outcome.MethodsPart I. The effects of different pneumoperitoneum on the peritoneal mesothelium and IL-1β、IL-6 in peritoneal fluid.SD rats were divided in ten groups. One group served as control. Pneumoperitoneum was established at 5 mmHg and 1.0 L/min of gas flow for 1 h, 2h and 3h with CO₂ （group C1h, C2h, C3h） and He （group H1h, H2h, H3h）. CO₂ pneumoperitoneum was further established at 8 mmHg and 1.0 L/min of gas flow for 1 h （group C8p）, at 5 mmHg and 2.0 L/min of gas flow for 1 h （group C2f）, at 5 mmHg and 3.0 L/min of gas flow for 1 h （group C3f）. After the procedures, five specimens were sampled from anterior peritoneum and measured through stereology and SEM. At the same time, the peritoneal fluid in groups CO₂ were collected, and ELISA immunoassay were adopted to evaluate the concentration of IL-1βand IL-6.Part II. Estimation of the injury in rat peritoneum after pneumoperitoneum vs laparotomy.SD rats were randomly divided into three groups. CO₂ pneumoperitoneum was established at 5 mmHg and 1.0 L/min of gas flow for 4 h. One group underwent laparotomy for 4h, and control group underwent anesthesia only. After the procedures, five specimens were sampled randomly from anterior peritoneum and quantitatively measured through stereology and SEM. Additionally, the peritoneal fluid was collected, and ELISA immunoassay were adopted to evaluate the concentration of IL-1βand IL-6.Part III. Application of stereology to compare the morphological effects after laparoscopy-assisted radical gastrectomy with that after open gastrectomy. 58 patients with gastric cancer in our center were selected as objects.Radical gastrectomy was performed for 31 cases by laparoscopy-assisted gastrectomy and 27 cases by conventional open gastrectomy. In both groups, the specimens were sampled randomly from anterior peritoneum after operation and quantitatively measured through stereology and SEM.Results（1） Group H1h and C1h, H2h and C2h, H3h and C3h, were both the same in the area fraction of the basal lamina exposed and diameter of mesothelial cells （P>0.05）. The magnitude of the peritoneal trauma in group C2h, C3h, C8p, C2f, C3f were significantly higher than that in group C1h （P<0.01）. And the same results were observed in group H2h and H3h against group H1h （P<0.01） and in group C3f against group C2f （P<0.01）. Furthermore, the area fraction of the basal lamina exposed in group C3h, H3h were remarkably higher than that in group C2h and H2h respectively （P<0.01）. The mechanism of the basal lamina exposed comprises the mesothelial cells’desquamating and plasmatorrhexis.（2） With the increasing of duration and gas ?ow of CO₂ pneumoperitoneum, the concentration of IL-1βand IL-6 in group C2h, C3h and C3f were higher than that in group C1h （P<0.05）. On the other hand, the concentration of IL-1βand IL-6 in peritoneal fluid didn’t change significantly when pressure was increased（P>0.05）.（3） Both CO₂ pneumoperitoneum and laparotomy caused peritoneal morphologic trauma. But there was no different between two groups （P>0.05）. Additionally, the mechanism of peritoneal trauma may be different between two groups.（4） Both CO₂ pneumoperitoneum and laparotomy caused peritoneal inflammatory reaction （P<0.01）. And the concentration of IL-1βin group CO₂ pneumoperitoneum was significantly lower than that in group laparotomy（P<0.01）. Meanwhile, the concentration of IL-6 in group CO₂ pneumoperitoneum was lower than that in group laparotomy （P<0.05）.（5） Both Laparoscopy-assisted radical gastrectomy and conventional open radical gastrectomy caused peritoneal morphologic trauma. And there were no different in the area fraction of the basal lamina exposed and the diameter of mesothelial cells between two groups （P>0.05）. Additionally, the mechanism of peritoneal trauma may be different between two groups.Conclusion（1） The peritoneal morphologic trauma during pneumoperitoneum shall be attributed to the pressure, duration and gas ?ow instead of the type of gas insufflated.（2） The peritoneal inflammatory reaction after CO₂ pneumoperitoneum shall be attributed to duration and gas flow instead of the pressure within the standard pneumoperitoneal working pressures.（3） The peritoneal morphologic trauma caused by prolonged CO₂ pneumoperitoneum was almost identical to that caused by laparotomy. And the peritoneal inflammatory reaction in group CO₂ pneumoperitoneum was lower than that in group laparotomy. Therefore, laparoscopic surgery seems to be suitable for abdominal malignancies.（4） Compaired with laparotomy, laparoscopic procedures may not only shows clinically relevant advantages such as better esthetic results and faster recovery, but also causes almost the same peritoneal morphologic trauma. Thus, laparoscopic surgery causes less effect on peritoneal metastasis of gastric cancer than laparotomy.