The Influrence Of Artificial Pneumoperitoneum On Piglets Circulation System, Respiration System, Peritoneum Morphology And Immunological Function

Recently, more and more diagnosis and treatment of disease in childhood can be solved by using laparoscopy, especially applying in the field of neonatal diseases. In addition to innovation of surgical instruments and operative method, surgical procedure is more complex, operative time is longer. And also there are an increasing number of children with preoperative complications. The influence of CO₂ pneumoperitoneum of this group has not yet fully understood. Effect of CO₂ pneumoperitoneum on respiratory, circulatory and metabolic area arouses people to search for the best filling gas constantly. Because of similar physical and chemical properties of N₂O to CO₂, N₂O becomes another focal point. A large number of clinical practices have shown the safety of N₂O applications. It is better than CO₂ in acid-base and blood flow dynamics, postoperative pain and so on. It has obvious advantages when appling in pregnant woman, heart and lung dysfunction patients. With the maturity of laparoscopy, it is gradually applied in pediatric oncology. However, it is still discussion intensely that whether CO₂ as a filling medium effect malignant tumor cells behavior and has potential impact to anti-tumor cells. Some scholars believe that this effect is caused by peritoneal damage because of applying CO₂ pneumoperitoneum.Therefore, this experiment established artificial CO₂ and N₂O pneumoperitoneum, in order to understand the influence of CO₂ and N₂O pneumoperitoneum on cardio-pulmonary function in piglets, compare advantages and shortcomings of different gas types, look into issues arising in neonates by using laparoscopy. By observation changes of peritoneal morphology under different gas exposition, investigation influence of CO₂ on metabolic and morphology changes in mesothelial cells and on transmigration of neuroblastoma cells across mesothelial cells, in order to discuss influence of CO₂ on immune of peritoneal cavity and explaining underlying mechanism of high rates tumor metastasis after lapascopy. Thus it can provide clue to discover the best filling gas when apply laparoscopy in neonates and tumor patients. PART 1A Research on The Influrence of Carbon Dioxide and Nitrous Oxide Pneumoperitoneum on The Circulatory, Respiratoryof PigletsObjective This experiment established pneumoperitoneum model and acute cardiac insufficiency model in piglets. It studies changes of hemodynamics and resiperitory system of artificial pneumoperitoneum with CO₂ and N₂O in piglets and in order to contrast superior and inferior of the filling gas for laparoscopy in neonate.Methods Thirty five piglets (2-4 kilogram in weight, 7-14 days of age) were equally divided into five groups: control group (CON), CO₂ group (CO₂), N₂O group (N₂O), cardiac failure & CO₂ group (CO₂-HF) and cardiac failure & N₂O group (N₂O-HF). It was going on 2h pneumoperitoneum in CON, CO₂, N₂O groups and 4h pneumoperitoneum in CO₂-HF, N₂O-HF groups. Investigation time points were 5min after anaesthesia and before pneumoperitoneum started, 15min, 30min, 60min, 120min and 60min after abdominal closure in CON, CO₂ and N₂O groups. Investigation time points were 5min after anaesthesia and before pneumoperitoneum started, 15min, 60min, 120min, 180min, 240min and 30min after abdominal closure in CO₂-HF and N₂O-HF groups. Investigation index were heart rete, stroke volume index, cardiac index, mean arterial pressure, central venous pressure, mean pulmonary arterial pressure, systemic vascular resistance index, dP/dtmax, partial pressure of carbon dioxide in arterial blood, partial pressure of oxygen in arterial blood, arterial oxygen saturation, concentration of HCO₃^- in arterial blood and blood PH value.Results Hemodynamics and parameters of respiratory were nearly constant in CON group. In CO₂ group, HR increased (P<0. 05), MPAP and SVRI increased (P<0. 001, P<0. 01), SVI decreased (P<0. 05), CI was stable. In N₂O group, HR decreased (P<0.001); CVP, MPAP and SVRI increased (P<0. 05, P<0. 001, P<0. 001); dP/dtmax, MAP, SVI and CI decreased (P<0.001, P<0. 001, P<0. 01, P<0.01). In CO₂-HF and N₂O-HF groups, compared to before modeling, when model was established, HR decreased (P<0. 01, P<0.01), SVI decreased (P<0. 05, P<0.01), CI decreased (P<0.001, P<0.001), MPAP increased (P<0.05, P <0. 05), SVRI increased (P<0. 01, P<0. 05), dP/dtmax, MAP and CVP were stable. Afterwards, HR were stable in both groups, CI decreased further (P<0. 05, P<0. 05) in both groups, CVP increased (P<0. 001, P<0. 05) in both groups, MPAP increased (P<0.01, P<0.05) in both groups. In CO₂-HF group, MAP decreased (P<0.001), SVI decreased further (P<0.05), SVRI increased further (P<0. 001), dP/dtmax decreased (P<0. 05).In CO₂ group, PaCO₂ increased (P<0. 001), accompanyed with PH value decreased (PH<0. 001). In N₂O group, PaCO₂ increased (P<0. 01). In CO₂-HF and N₂O-HF groups, compare to before modeling, when model was established, PH value decreased (P<0.01, P<0.001), PaCO₂, PaO₂, SaO₂ and HCO₃^- were stable. Afterwards, PaCO₂ increased (P<0. 05, P<0.001) in both groups, PaO₂ (P< 0.05, P<0. 001) and SaO₂ decreased (P<0. 05, P<0. 01), HC0₃^- decreased (P< 0.01, P<0. 01), PH value decreased (P<0. 05, P<0. 001).Conclusion The experimental results suggested that healthy piglets can tolerate CO₂ pneumoperitoneum and CO₂ pneumoperitoneum was superior to N₂0 pneumoperitoneum. Cardiac contractility decreased and systemic vascular resistance index increased obviously when applying CO₂ pneumoperitoneum in cardiac insufficient piglets for long duration. Long duration of N₂0 pneumoperitoneum has no further myocardial damage to cardiac insufficient piglets. PART 2A Research on The Influence of Carbon dioxide on Morphology of Peritoneum and Immunologic functionObjectiveThe response of mesothelial cells, one of the dominant cell population in the peritoneal cavity, to surgical trauma is poorly defined. Nonetheless, electron microscopic studies have shown significant morphologic changes of these cells after CO₂ pneumoperitoneum in vivo. It is recently shown that CO₂ pneumoperitoneum increases systemic metastasis of peritoneal neuroblastoma cells in a murine model. Thus, this experiment was tested the effection under different gas condition to the morphology and the inflammatory response of peritoneal mesothelial cells and discussed the underlying mechanism of the effection of CO₂ pneumoperitoneum to tumor cells in peritoneal cavity in vitro.MethodsPurification of primary murine peritoneal mesothelial cells was achieved by sequential peritoneal lavage after 0. 125% trypsin pretreatment. Purity of the mesothelial cell culture was confirmed by cell morphology and immunohistochemical staining for specific cytokeratine (AE1/AE3). In all experiments vitality of the cells was confirmed by MTT assay. Cells were stimulated by LPS (10μg/ml) or murine TNF-α(10ng/ml) and the release of G-CSF, IL-6, M-CSF, MCP-1 and RANTES during and following CO₂ incubation were measured by ELISA. In an additional set of experiments monolayers of mesothelial cells were established on transwell systems. Following incubation with 100%CO₂ or 5%CO₂ for 2h, fluorescent stained Neuro2a cells were added to the upper chamber and their migration into the lower chamber was measured by multi-detection reader. Peritoneum and isolated mesothelial cells were detected by scanning electron microscopy.Results MTT conversion was decreased during the CO₂ incubation both in LPS and TNF-αstimulation groups. This inhibition effect was longer in TNF-αgroup than in LPS stimulation group. After 24h, their mitochondrial activity recovered. Mesothelial cells stimulated with LPS or TNF-αreleased significant amounts of IL-6 and G-CSF (P<0. 05). The production of M-CSF, MCP-1 and RANTES can not be stimulated obviously. The release of G-CSF and IL-6 was completely blocked during the incubation with 100%CO₂ (P<0. 05), but directly after the incubation the inflammatory capacity recoverd. M-CSF can not be effected by CO₂ obviously. Migration studies showed that the barrier function of the mesothelial monolayer decreased. A significantly increased migration of neuroblastoma cells was identified after 100%CO₂ exposure (P<0. 05). Scanning electron microscopy investigation suggested 100%CO₂ exposition was associated with a significant destruction of the microvilli formation of isolated mesothelial cells in vitro. In vivo, it also suggested 12mmHg 100%CO₂ pneumoperitoneum destroyed mesothelial cells layer of peritoneum when lasting for 4h, basal lamina was exposed and cytoskeleton was existed merely. In contrast, 100%N₂O pneumoperitoneum leaded to increase of intercellular gaps and basal lamina was exposed in part areas under same pressure and duration.Conclusions100%CO₂ suppressed mitochondrial activity of mesothelial cells temporarily. Peritoneal mesothelial cells released significant amounts of cytokines following stimulation. This inflammatory response was blocked during the incubation with 100%C0₂, but there appeared to be no lasting effect. Moreover the peritoneal mesothelial cells lose their typical cell morphology by 100%CO₂ exposition, which is accompanied by facilitated migration of neuroblastoma cells. Thus, the increased Neuroblastoma metastasis observed after CO₂ pneumoperitoneum in mice might be related to an impaired mesothelial barrier function.